The spread and antibiotic resistance of Salmonella enterica serotype typhimurium DT104 in Hungary.

Comparison of phage types (PTs) determined by Felix and Callow's and Anderson's methods was performed testing 99 human strains of S. enterica serotype Typhimurium (S. typhimurium) isolated in Hungary. PT2 and PT2c--according to Felix-Callow--corresponded with Anderson's DT104 in case of 39 strains out of 40. Among 59 isolates belonging to other Felix-Callow's PTs only one strain was found which was DT 104. Similar unambiguous equalities could not be established between any other PTs comparing the two methods. The PTs of 17,877 human strains isolated between 1988 and 1999 were determined using Felix-Callow's method. On the basis of the above equality the emergence of DT104 could be followed retrospectively by means of the rate of PT2 and PT2c. The increase of DT104 began already in 1989, emerging first PT2c then PT2. It predominated since 1991 and it reached its maximum (78.3%) in 1999. The incidence of multiresistance among one of the groups of DT104 strains (Felix-Callow's PT2) was significantly higher in 1998 than the average of non-DT104 strains. The predominant R-type was ACST.

Epidemiology and characterization of animal Salmonella enterica subspecies Enterica serotype typhimurium DT104 in Hungary.

Reports on the internationally emerging significance of multiresistant zoonotic Salmonella in animals and man prompted studies to estimate the significance of multiresistant Salmonella enterica subspecies enterica serotype Typhimurium (S. Typhimurium) phage type DT104 of animal origin in Hungary. A collection of 231 strains (primarily of goose, turkey, poultry and porcine origin from the years 1997-1998) was tested for resistance against 7 selected antibiotics (ampicillin, chloramphenicol, enrofloxacin, nalidixic acid, streptomycin, tetracycline and sulphamethoxazole). Strains with resistance against 3 or more were defined as multiresistant. All strains were phage typed using Felix-Callow's S. Typhimurium phage typing system, and 91 of them (suspect DT104) were also typed according to Anderson's definitive typing (DT) system. In this study, 14% of animal strains from 1997-1998 was classified as DT104, for which turkey, pig and duck seemed to be the main carriers, and the multiresistant non-DT104 strains represented a further 6% of this collection. The prevalence of DT104 was highest among strains of turkey origin (50%), followed by strains of pig (29%), chicken (25%), duck (19%), and goose (3%) origin. The other DT104 related phage types (DT12 and U302) were only detected in the case of 4 strains (2 of porcine, and one each of turkey and of goose origin). The DT104 corresponded to the Felix-Callow types 2/3 or 2c/3 in each case, except in the case of 3 turkey strains where they corresponded to type 35/3. Nalidixic acid resistance was detected in all multiresistant turkey strains and in some of other animal origin but none of these strains were resistant to enrofloxacin. A retrospective analysis (based on the above relationship) indicated that S. Typhimurium strains corresponding to DT104 could be present and increase in the Hungarian farm animal population from about 2% to 20% between 1985 and 1990, in a manner similar to the emergence of human DT104, as reported elsewhere (Pászti et al., 2000). The 91 suspect DT104 strains were also tested for plasmid profile and for spvC gene indicating the presence of the large serotype specific plasmid (Ssp). No characteristic plasmid profile could be attributed to S. Typhimurium DT104. The serovar-specific large plasmid was detected by PCR for spvC in 100% of DT104 strains and in 77% of the non-DT104 strains. The virulence of two DT104 strains was tested in orally infected day-old chicks and compared with virulence of 4 non-DT104 strains. Higher colonizing virulence of DT104 strains could be established as compared to the other strains.

Phage restriction and the presence of small plasmids in Salmonella enteritidis.

Between 1990-1994, a total of 16,505 S. enteritidis strains of human, animal and food origin were phage-typed, using the Hungarian scheme and the changes of incidence of the dominant phage types were monitored. The incidence of PT1 (corresponding to Ward's PT1 was very high between 1990 and 1992 (67.9-71.0% of the total S. enteritidis isolates), later, it decreased. The prevalence of PT6 (corresponding to Ward's PT4) was rare until 1992, then it gradually increased. The phage type and plasmid content of 78 Salmonella enteritidis strains were determined. Small plasmids were present in 59% of the isolates, together with a serotype-specific (38 MDa) plasmid. A correlation was found between the presence of the small plasmid and phage restriction to two phages used for subdividing the Hungarian phage types 1 (PT1) and 6 (PT6) of S. enteritidis (corresponding to PT1 and PT4 in Ward's typing scheme, respectively).

Effect of precultivation conditions on colicin susceptibility in Escherichia coli.

The effect of 20 colicins and cloacin was studied after various precultivations. Nutrient agar supplemented with subinhibitory concentration of EDTA used for precultivation or elevating the growth-temperature of the inoculum from 37 degrees C to 42 degrees C increased the susceptibility of wild-type (smooth) Escherichia coli strains to the inhibitory action of some colicins. There were great differences among the colicins in respect to these effects. In case of rough mutants, their sensitivities did not change or eventually decrease after EDTA or heat pretreatment. The LPS pattern in SDS-PAGE of smooth cells grown in EDTA-containing nutrient medium changed in some degree towards the rough character. In case of precultivation at 42 degrees C this change was less considerable. It is supposed that both factors applied during precultivation have influence on colicin sensitivity by means of the change of receptor activity caused by LPS modification.

Comparison of two Salmonella enteritidis phage typing methods.

In the last 10 years several phage typing methods were developed for Salmonella enteritidis, leading to confusion in the predominant phage types (PT) reported from different countries. We made comparative examinations on 1487 S. enteritidis strains isolated in Hungary in 1990-1991, using two phage-sets: a modified version of the method elaborated by László et al. (here in after Hungarian method) and the system of Ward et al. (here in after Colindale method). Typability of the strains was nearly the same: 98.0% and 98.3%, the isolates belonging to 18 and 19 phage types, respectively. The Hungarian method revealed 6 (1, 1c, 1b, 1d, 7, 18), the Colindale method 5 (1, 6, 8, 21, 26) frequent phage types. In Hungary PT 1 has been predominant since 1981 and using the Colindale method 64% belonged to this type; using the modified Hungarian method this type could be divided into PT 1, PT 1c, PT 1b and PT 1d. Other frequent phage types (PT 18, PT 7) were nearly identical with Colindale types PT 26 and PT 21.

Epidemiological and microbiological data on Salmonella enteritidis.

The number of Salmonella enteritidis isolations started to rise in humans, eggs and egg products in 4 territories out of the examined 9 territories of Russia in 1986. The spread of S. enteritidis infections was connected with the consumption of hen's eggs as it was demonstrated by the analysis of the local outbreaks. Phage type of 1142 S. enteritidis strains isolated in Russia was determined using the Hungarian typing scheme. The strains were typable in 95.3% and 12 phage types were found. Phage type 1 was the most frequent (86.7%) among human strains and also among strains originated from hen and egg products. The examined 18226 human S. enteritidis strains isolated in Hungary between 1984 and 1989 belonged to 24 phage types and phage type 1 was predominant, the incidence of this type varying between 69.3% and 93.2%. The strains were sensitive to antibiotics, multiresistant strains were found in 1%. Plasmid content was examined of 138 strains; a 38 Md plasmid was carried by all of them and a 96 Md plasmid was harboured by 11 antibiotic-resistant strains. The tested strains produced enterobactin but no aerobactin.

Correlation between human lactoferrin binding and colicin susceptibility in Escherichia coli.

Escherichia coli H10407 demonstrated low 125I-human lactoferrin (HLf) binding (7%) and was insusceptible to group A (A, E1, E2, E3, E6, and K) and group B (B, D, Ia, Ib, and V) colicins. Conversely, a spontaneous HLf high-binding (44%) variant, H10407(Lf), demonstrated an increase susceptibility to both colicin groups. Colicin-insusceptible E. coli wild-type strains 75ColT, 84ColT, and 981ColT showed a low degree of HLf binding, i.e., 4, 8, and 10%, respectively. The HLf binding capacity was high in the corresponding colicin-susceptible mutants 75ColS (43%), 84ColS (32%), and 981ColS (43%). Furthermore, HLf low- (less than 5%) and high- (greater than 35%) binding E. coli clinical isolates (10 in each category) were tested for susceptibility against 11 colicins. Colicin V susceptibility did not correlate with HLf binding in either categories. However, with the remaining colicins, three distinct HLf-binding, colicin susceptibility patterns were observed; (i) 10 of 10 HLf low-binding strains were colicin insusceptible, (ii) 6 of 10 HLf high-binding strains were also colicin insusceptible, and (iii) the remaining HLf high binders were highly colicin susceptible. Certain proteins in the cell envelope and outer membrane of wild-type H10407 (HLf low binder, colicin insusceptible) showed a lower mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared to the corresponding proteins of mutant H10407(Lf) (HLf high binder, colicin susceptible). These mobility differences were also associated with HLf-binding proteins in Western blot (ligand blot) analysis. The wild type showed a smooth form of lipopolysaccharide (LPS) with a distinct ladder of O-chains, compared to the rough LPS of the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiology of Salmonella derby strains isolated from swine, pork and pork products.

A total of 35 Salmonella derby strains, isolated from 6 types of samples of porcine origin from 9 different places in Hungary were examined for their characteristics. Thirty-two strains (91%) were of phage type 25, 2 (6%) of phage type 15 and 1 (3%) of phage type 58. Colicin production was observed in 3 (9%) strains. Five strains (14%) were found to be resistant to tetracycline (Tc). The strains harboured plasmids of 2.2, 2.4, 3.4, 4.2 and 72 Md. The 72 Md plasmid appears to be characteristic of S. derby and possibly encodes Tc resistance. The 72 Md plasmid belonged partly to incompatibility (Inc) group I1, while the other plasmid of the same size belonged to Inc. group B. The findings suggest that healthy salmonella carrier pigs carried the infection from the farm to the abattoir. Slaughtering of infected pigs may have led to contamination of the carcasses and, thereafter, that of the pork and pork products.

Subdivision of common Salmonella serotypes: phage typing of S. virchow, S. manhattan, S. thompson, S. oranienburg and S. bareilly.

The phage typing system elaborated for Salmonella infantis proved suitable to subclassify 633 S. virchow, 206 S. manhattan, 103 S. thompson, 30 s. oranienburg and 58 S. barielly strains. The S. virchow strains belonged to phage type 213 in 79.6%, each of six phage types occurred more frequently than 1%, and 15 phage types less frequently than 1%. The S. manhattan strains belonged to three phage types (687, 247, 547) in 91.2% and nine other types were found. The most common phage types among the S. thompson strains were 243 and 247 (52.4 and 18.4%) and eleven other types were encountered. All S. oranienburg strains derived from an outbreak and belonged uniformly to phage type 111. S. barielly strains isolated from an other outbreak fell into phage type 683.

A phage typing system for Salmonella infantis.

A phage typing method applying 9 type phages was elaborated to subdivide Salmonella infantis. Results are reported by the use of Farmer's mnemonic. Out of 4847 S. infantis strains, 4602 were of human and 245 of non-human origin. The strains were typable in 98.9%. Two phage types occurred more frequently than 20%, four phage types between 5 and 10%, seven phage types less than 5%, and twenty-eight phage types less than 1%. The strains originated from outbreaks in 28.7% and from sporadic cases in 71.3%. A total of 1320 strains examined for phage type was isolated from 4 field epidemics, 39 community outbreaks and 370 family infections. In the second version of the method two phages were substituted by two more effective ones. The phage typing method was suitable for epidemiological purposes. Inducing in vitro changes in phage types by lysogenization and plasmid acquisition, phage types 111, 113, 311, 313 and 343 changed to phage types 213, 243, 513 and 543 after lysogenization and phage types 311 and 543 to phage types 548, 565 and 885 due to plasmid acquisition.

Antibiotic resistance and plasmid profiles of Escherichia coli and Klebsiella isolated from in-patients receiving prolonged antibiotic therapy.

Distribution by serogroup, phage type, colicin production, colicin type, sensitivity to antibiotics and plasmid characteristics of 74 Escherichia coli and 11 Klebsiella strains isolated from hospitalized patients receiving prolonged antibiotic therapy indicated that the infections were not associated with the hospital environment. Resistance was tested to 26 antibiotics, some of them being not generally used in therapy; 30 strains were resistant to 4 to 17 antibiotics. There was a significant difference in the antibiotic resistance of strains derived from patients with urinary-tract infections (UTI) and with leukaemia (LP). As compared to the UTI group, among E. coli strains in the LP group the frequency of multiple resistance was significantly higher, the MIC values were higher and R-plasmids were more frequent. Out of 30 multiple resistant E. coli strains 27 were R-plasmid carriers. Three different kinds of plasmid profile were shown in more than one strain (2 out of 10 UTI strains and 3 and 2 out of 10 LP strains). The rest of the isolates differed in plasmid profile from these and from one another; the presence of "epidemic plasmid" was not demonstrated. Plasmid epidemiological examinations may forecast the efficacy of an antibiotic or of a group of antibiotics.

Epidemiological analysis of Salmonella typhi-murium infections on the basis of laboratory methods. I. Distribution of phage types and biotypes of Salmonella typhi-murium isolated in Hungary in the period 1960 to 1981.

Phage and biochemical types were determined of 34 937 Salmonella typhi-murium cultures including 31 708 human strains, 2732 animal strains and 497 strains isolated from water. Phage type 4, not typable strains (nt) and phage type 2b were predominant among the strains of human and animal origin, and nt, 4 and 2b among the strains isolated from water. The most frequent phage types and the nt strains were subdivided by biotyping and additional phages. The incidence of S. typhi-murium var. copenhagen strains was 12.4%, they belonged mainly to phage types 2b and nt. The number of S. typhi-murium isolates of human origin showed a 2-4 year periodical fluctuation between 1960 and 1981. A connection was found between the incidence of the predominant phage types (4, nt, 2b) and the periodical changes in the total number of isolates. Phage type 4, which predominated among the strains of human and animal origin till 1976, was ousted gradually by nt ones. In the period when the change in predominance was observed the number of epidemics decreased and the number of sporadic cases increased. The change in the frequency of phage types took place at the same time when the frequency of phage types changed among the strains isolated from cattle and meat-products (4----nt). The increased number of sporadic cases after 1976 refers to infections from cattle and not from poultry.

Epidemiological analysis of Salmonella typhi-murium infections on the basis of laboratory methods. II. Resistance to antibiotics and R-plasmid carrier state in Salmonella typhi-murium isolated in Hungary in the period 1974 to 1981.

Phage and biochemical typing of Salmonella typhi-murium strains performed in the course of the Salmonella Surveillance Programme were completed with examinations on resistance to antibiotics and R-plasmids. A total of 15 600 strains of human, animal and water origin were tested between 1975 and 1981 and most of the monoresistant strains were found among the animal strains (73.6%-94.9%), while double resistance was the most frequent among the human strains (5.5%-25.5%) and multiresistance occurred in the highest ratio (2.8%-25.6%) among the strains of water origin. Tetracycline resistance was the most frequent in all the three materials. The curves representing the incidence of tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), kanamycin (Km) resistance were similar to the curve of multiple resistance and differed from the curve showing the incidence of ampicillin (Ap) resistance. Gentamicin resistance was found only among human strains (in 0.3 and 0.7%), strains resistant to nitrofurantoin and co-trimoxazole occurred among strains of human and water origin in low percentages. The most common antibiotic resistance patterns of the multiple resistant strains were Tc, Cm, Sm, Km, Ap, Su; Tc, Sm, Su; Sm, Km, Ap, Su. Multiple resistant strains belonged in the majority to phage type nt (not typable) and 2b. Out of the examined 512 S. typhi-murium strains resistant to antibiotics, the presence of R-plasmid was demonstrated in 408 strains (i.e. 79.7%). The R-plasmids, derived from strains of human, animal and water origin, of phage type nt, biotype 3 (nt/3) isolated in 1979, were characterized according to the resistance determinants, fi-character, incompatibility-group, phage-inhibition and molecular weight. Two kinds of R-plasmids were carried by three human strains (FI and H, FI and alpha). R-plasmids belonging to Inc P and Inc H were carried by one animal strain. Strains isolated from sewage carried R-plasmids of Inc groups H and I alpha. Out of the examined 15 S. typhi-murium strains of phage type 2b, isolated in 1981, the molecular weight for 7 strains was 66 Md and four belonged to Inc I alpha. The R-plasmids derived from 2b/2 strains, isolated in the same county, were identical according to antibiotic resistance determinants, phage inhibition and molecular weight. The molecular weights of R-plasmids derived from 10 strains out of the examined 30 nt strains were also 66 Md and the four examined plasmids belonged also to Inc I alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Phage types and epidemiological significance of Salmonella enteritidis strains in Hungary between 1976 and 1983.

In Hungary, 14 819 human Salmonella enteritidis strains were isolated between 1976 and 1983. Phage type was determined of 10 132 human strains originating from 6852 foci, and of 711 strains isolated from animals and water in this period. The human strains were typable in 99.4% and they belonged into 21 phage types. Five phage types (1, 4, 7, 16 and 17) were more frequent than 1%. Phage type 7 predominated among the strains isolated between 1976 and 1980, including 65.6%-89.3% of the strains. There was a change in the prevalence of phage types from 1980-1981, as phage type 7 was ousted by phage type 1. The date of the change in the predominance of phage types coincided with the considerable increase of S. enteritidis isolates; the number of isolates was nearly fivefold in 1980 of that in 1976. Phage type 7 frequent in the first period proved to be homogeneous; the strains could not be subdivided either by the temperate phages carried by them or by other phages. The incidence of phage types 1 and 7 was nearly the same among the strains derived from animals, food, water and hygienic control examinations, and there was no temporal difference in the frequency of the two phage types as it was observed among the human strains. The human strains originated in 49.5% from outbreaks and in 50.5% from sporadic cases in the country. Of the strains examined for phage type during the eight-year period, 41.9% were isolated from 23 field epidemics, 84 community outbreaks and 757 family infections. Analysing the regional spread of S. enteritidis, the increase in the number of isolates was the highest in counties Tolna, Bács-Kiskun, Somogy and Györ-Sopron. The predominance of phage type 1 was observed in counties Békés, Borsod, Csongrád, Györ-Sopron, Hajdu-Bihar, Pest and Tolna. It was obvious in the case of county Tolna that the source of infection was contaminated egg and baby chicken. Phage type 7 predominated in counties Komárom, Vas and Veszprém. Phage type 4 circulated in counties Csongrád and Pest, phage type 17 in county Fejér and phage type 2 in county Hajdú-Bihar.

Change in phage-sensitivity of Shigella flexneri strains. I. Changes in virulence and phage-sensitivity.

In the course of repeated passaging of virulent Shigella flexneri cultures in liquid media, avirulent variants and variants of reduced virulence appeared. They were easily recognizable by their colonial morphology. The avirulent variants became sensitive to certain phages not lysing the original virulent strains. The change of phage-sensitivity among the variants with no or decreased virulence was significant: chi 2 = 64.25; P less than 0.001. The amplification of phage-sensitivity developed in 15% of the originally avirulent cultures. No association was found between the loss of virulence and a specific phage-sensitivity pattern. In the majority of the cases sensitivity to phage Ms2 developed during the passages, but it was observed in some of the original strains, too. With few exceptions, sensitivity to phage Ms2 was associated with the total or partial degradation of the specific antigen and the antigenic structure detectable by S. flexneri factor sera. These exceptions were cultures with maintained or decreased virulence.

Changes in phage-sensitivity of Shigella flexneri strains. II. The F-like character of Ms2-sensitive variants.

Variants sensitive to male specific phage Ms2 appeared among Ms2 non-sensitive Shigella flexneri cultures in the course of liquid medium passages. Sensitivity to Ms2 was lost on acridine orange treatment and was transferable into Ms2 non-sensitive variants. The Ms2 sensitive variants had fimbrial antigen. Electron microscopy showed that Ms2 phages were adsorbed on the F-like fimbriae of these variants. It was assumed that F-like plasmids determining F-like fimbrial antigen were carried by these variants.

Salmonella and shigella surveillance in Hungary, 1972-1976. II. Shigella surveillance.

Shigellae were isolated from 32 399 persons in the bacteriological laboratories of the Public Health Stations between 1972-1976. The number of isolations ranged between 5325 and 8237 yearly. As S. dysenteriae and S. boydii serotypes occurred only in about 1.5% of all isolations, the epidemiological situation was determined by the incidence of S. flexneri and S. sonnei. Except in 1973, S. sonnei constituted the majority in every year. S. sonnei predominance, observed first in the western regions of the country, showed a gradual eastward spread and became characteristic of all localities in 1975-1976. The July-September peak of shigella isolations was associated with S. sonnei, as S. flexneri was distributed practically evenly in every season. The incidence of shigella isolations per 100 000 inhabitants was the lowest in the middle and the highest in the northern parts of the country. Sporadic cases were somewhat more frequent than epidemic outbreaks. Shigellae were isolated in 64.1% from patients and in 35.9% from asymptomatic excreters. The patient : excreter ratio was higher for S. sonnei (70.2 : 29.8) than for S. flexneri (54.6 : 45.4). In the survey period, 14 692 isolations were made in the course of 3278 epidemics; out of these, 11 171 cases were involved in 574 extensive outbreaks. S. sonnei was responsible mainly for school and nursery outbreaks, whereas S. flexneri dysentery predominated in mental wards and in homes for the aged and for mentally retarded children. The prevalent types of S. flexneri were (serological/phage type): 2a/69, 3a/5, 3a/19, 4a/81, 4a/89d and 6/85. The prevalent epidemiological subunits of S. sonnei were (phage/colicin type): 2/0, 2/6, 2/12, 3/12, 6/0, 7/0, 65/0, 65/6 and 65/12.

Salmonella typhi R-plasmids in Hungary.

The antibiotic sensitivity of 4095 Salmonella typhi strains isolated in Hungary from January, 1974 to June 1979, was tested. Twelve strains derived from one patient and seven chronic carriers were resistant to antibiotics due to R-plasmids. One of the S. typhi strains carried two R-plasmids and the change in the phage type of this strain was caused by one of the plasmids. R-plasmid of the same restrictive property was found in an Escherichia coli strain isolated from the faeces of the corresponding carrier. All of the R-plasmids examined were fi-, E. coli and Shigella flexneri phages were restricted by four R-plasmids, S. typhi phages were restricted by one. R-plasmids belonged to incompatibility groups I1, I2, W and H.

R factors derived from Shigella flexneri strains.

Of 2492 Shigelle flexneri strains isolated from dysenteric patients in Hungary in the years 1972-1974, 767 (30.8%) were resistant to 1-5 antibiotics. Resistance was due to R factors in 79.2% of the strains. Fertility inhibition experiments with F specific phages showed R factors to be fi+ in 12.9% and fi- and 87.1%. Of the antibiotic markers, the chloramphenicol-tetracycline resistance determinant was the most common (46.4%). Chloramphenicol resistance determinants were carried out by fi- R factors.

Restriction and modification of Shigella flexneri phages by R factors.

Out of 420 R factors derived from Shigella flexneri strains, 50.8% restricted Escherichia coli and S. flexneri phages. Phage restriction was produced both by fi- and fi+ R factors. The R factors were divided into nine groups on the basis of the efficiency of plating of S. flexneri phages. Changes of phage types were produced by transferring R factors of different restrictive types. The changes offered some information concerning the evolution of phage types. Studies on phage modification supported the grouping of R factors determined on the basis of restriction. R factors of different restrictive types were type-specific except for types VII and IX. Modified phages proved to be highly practical for epidemiological purposes. The use of modified phages, as an additional phage-set besides that basic phage-set, was suggested to trace the source of strains which changed their phage types as an effect of R factors.