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.

Phenotypic and genotypic properties of methicillin resistant Staphylococcus aureus strains isolated in Hungary, 1997-2000.

An account is given on the activity of the National Center for Phage Typing of Staphylococci in Hungary in the period between 1997 and 2000 related to methicillin resistant Staphylococcus aureus (MRSA) strains originating mainly from hospital infections and sporadic cases. The rate of multiresistant MRSA strains has decreased gradually from 98.1% in 1997 to 74.6% in 2000, accordingly the typability by phages showed a considerable improvement by the international basic phages. Resistance pattern of MRSA strains became narrower in the period of the examinations. With the exception of erythromycin the rate of resistance decreased probably as a consequence of the increased use of erythromycin. The typing method was completed with the phenotypic and genotypic characterization of macrolide resistance. Among 73 MRSA strains type A was the most frequent macrolide resistance group, while type B, C1 and C2 occurred rarely. Type A was frequent also among the few MSSA and CNS strains. Out of the 168 examined S. aureus strains ermA genes occurred in 81.5%; in MSSA and CNS strains ermC1 genes were frequent, both genes are responsible for the target modification. The msrA gene, encoding the increased efflux, occurred only in CNS strains. Comparing the results obtained by phenotyping (phage typing) and genotyping (AP-PCR) methods it is of note that MRSA strains which proved non-typable by phage typing gave suitable results by the AP-PCR.

First searchable database for DNA profiles of human cell lines: sequential use of fingerprint techniques for authentication.

The authenticity and freedom from cross-contaminants of a cell line are important prerequisites for any research, development or production programs involving cell lines. Mini- and microsatellites in the human genome harboring variable-numbers of tandem repeat (VNTR) DNA markers allow individualization at the DNA level and are of practical value for genetic linkage mapping, forensic legal medicine, paternity testing, monitoring of bone marrow transplants, and individualization of established cell lines. We have validated fingerprint techniques of different single- and multiple-locus VNTRs enabling the establishment of a searchable database of DNA profiles. As a result, multiplexed polymerase chain reaction amplification fragment length polymorphism (AmpFLP) of four prominent and highly polymorphic minisatellite VNTR loci was proven as the best tool for screening the uniqueness of DNA profiles in a fingerprint database. In order to avoid false positivity, identical or similar DNA profiles based on AmpFLP VNTR were tested further using a multi-locus fingerprint system. Our data demonstrate that misidentification remains a chronic problem among human continuous cell lines (detailed information at URL http://www.dsmz.de). The combination of rapidly generated DNA profiles based on single-locus VNTR loci, their authentication by screening the fingerprint database, and confirmation of duplicate banding patterns using multilocus fingerprints constitute a highly reliable and robust method, which enables high fidelity and quality of maintenance independent from the quantity of individual cell lines.

Spread of a Salmonella typhimurium clone resistant to expanded-spectrum cephalosporins in three European countries.

Twelve Salmonella typhimurium strains resistant to broad-spectrum cephalosporins were isolated from cases of gastroenteritis during 1996 to 1998 in Russia, Hungary, and Greece. Resistance was due to the production of CTX-M-type extended-spectrum beta-lactamases encoded by similar 12-kb plasmids. By pulsed-field gel electrophoresis, all strains shared the same chromosomal type. These data suggest that an S. typhimurium clone resistant to broad-spectrum cephalosporins is present in at least three European countries.

Widespread intraspecies cross-contamination of human tumor cell lines arising at source.

We present a panoptic survey of cell line cross-contamination (CLCC) among original stocks of human cell lines, investigated using molecular genetic methods. The survey comprised 252 consecutive human cell lines, almost exclusively tumor-derived, submitted by their originators to the DSMZ and 5 additional cell repositories (CRs), using a combination of DNA profiling (4-locus minisatellite and multilocus microsatellite probes) and molecular cytogenetics, exploiting an interactive database (http://www.dsmz.de/). Widespread high levels of cross-contaminants (CCs) were uncovered, affecting 45 cell lines (18%) supplied by 27 of 93 originators (29%). Unlike previous reports, most CCs (42/45) occurred intraspecies, a discrepancy attributable to improved detection of the more insidious intraspecies CCs afforded by molecular methods. The most prolific CCs were classic tumor cell lines, the numbers of CCs they caused being as follows: HeLa (n = 11), T-24 (n = 4), SK-HEP-1 (n = 4), U-937 (n = 4) and HT-29 (n = 3). All 5 supposed instances of spontaneous immortalization of normal cells were spurious, due to CLCC, including ECV304, the most cited human endothelial cell line. Although high, our figure for CCs at the source sets a lower limit only as (i) many older tumor cell lines were unavailable for comparison and (ii) circulating cell lines are often obtained indirectly, rather than via originators or CRs. The misidentified cell lines reported here have already been unwittingly used in several hundreds of potentially misleading reports, including use as inappropriate tumor models and subclones masquerading as independent replicates. We believe these findings indicate a grave and chronic problem demanding radical measures, to include extra controls over cell line authentication, provenance and availability.

Partial inhibition of amplifications by primers of EHEC genes.

Diagnostic value of multiplex polymerase chain reaction (PCR) was examined by using three primer pairs, specific for the common conserved region of stx1 and stx2, eae and an enterohaemolysin A gene (ehxA). The sensitivity in respect of each amplicon decreased with three exponents comparing to the individual PCR reactions. These PCR reactions were partially inhibited by the presence of certain additional primers. This inhibitory effect was template-concentration dependent, and was partially balanced by usage of increased amount of dNTP. Taq DNA polymerase in a range of 0.3-1.25 U/reaction did not influence the inhibition. The same inhibition was detected if the annealing temperature was changed from 48 degrees C to 57 degrees C. Pairs of EHEC primers inhibited a Salmonella enteritidis virulence-plasmid specific gene amplification, as well. Theoretical inhibiting effects were predicted by Primer Premier software but our observations can be sufficiently explained neither by the competitions between the specific and aspecific amplifications nor by the inhibition caused by dimerization of primers.

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).

Advance in bacterial typing methods (a review).

We give a short review about the two main groups of bacterial typing methods for epidemiological purposes: the phenotypic and genotypic methods. The advantage of the phenotypic methods is their feasibility for the less equipped laboratories, their disadvantages are the variability of the phenotypic properties, and their high labor requirement. The advantages of the genotypic typing methods are the higher discriminatory power and applicability of the same method for different species of bacteria. The disadvantage of these methods is that they are expensive and labour consuming. Beside the short description, we show the results of different authors obtained by the use of the molecular methods in the epidemiological practice and we call the attention to the insufficiency concerning the stability.

Detection of VTEC using specific DNA probes and complex typing of Escherichia coli O157.

The incidence of E. coli causing hemorrhagic colitis (HC) or non-bloody enteritis in Hungary was studied using SLT-I and SLT-II gene-probes as well as Vero-cell toxicity and Verotox-F tests. Out of 41 E. coli O157 strains isolated in Hungary between 1987 and 1996 15 strains (O157:HNM 4, O157:H77 8, O157:HNT 3) derived from hemorrhagic colitis (HC). Hybridization was observed with SLT-I and/or SLT-II in 19 strains. Verocytotoxin production of E. coli of 23 other serotypes was proven by hybridization of DNA probes. SLT production were demonstrated in 24 strains. Complex typing (sero-, phage-, colicin-typing and plasmid profile analysis) was carried out in E. coli serogroup O157 strains isolated from different geographical areas. Using the Hungarian phages the E. coli O157:HNM, O157-H7 strains could be distributed into 6 phage groups each and these phage groups could be further divided according to colicin production and plasmid profile. The Hungarian phage typing method for E. coli strains used since 1978 was compared to the method elaborated in Canada in 1990. Out of the most frequent Canadian phage types (1, 4, 8, 31, 14) phage types 8, 31 and 14 were observed in Hungary.

Examination of verocytotoxin producing capacity and determination of the presence of Shiga-like toxin genes in human Escherichia coli isolates.

A total of 93 wild type Escherichia coli of human origin (mostly representing intestinal isolates from Hungary) were examined for the presence of Shiga-like-toxin (SLT) genes using SLT-I, SLT-II and enterohemorrhagic E. coli (EHEC) specific DNA probes. The structural genes of the above specificity were labelled by random priming using 32-P-dCTP. E. coli strains investigated represented 16 serogroups: O1, O2, O4, O5, O6, O18, O25, O26, O45, O55, O111, O125, O126, O128, O157, O165, members of which were likely to produce verotoxin (VT) and strains of serotypes O11:NM, ONT:NM isolated from six haemorrhagic uraemic syndrome (HUS) patients. Out of these strains 51 were examined for in vitro VT production capacity. Only one strain (an O26:H11 from Germany) produced VT. This was also the only strain which proved to be positive with one of the SLT probes (SLT-II), and with the EHEC probe. From this strain a phage was isolated which was proven to be nonconvertible. These data support epidemiological and clinical observations about the very low occurrence or absence of EHEC in Hungary, in contrast to many European countries.

Genotypic and phenotypic characters and nosocomial significance of bacteria endemic in neonatal intensive care units.

The degree of colonization was determined by complex typing (sero-, phage, colicin-, pyocin typing, plasmid profile analysis) of 212 Escherichia coli, 232 Klebsiella, 117 Pseudomonas aeruginosa and 52 Staphylococcus aureus strains isolated from nose, throat, ear and other sources of 563 new-born infants in gynaecological and maternity wards of two neonatal intensive care units (NICU I and II) during a one year period. The presence of Klebsiella strains was more frequent in NICU I and E. coli and P. aeruginosa in NICU II, S. aureus occurred in a low level in both units. In NICU I 34 kinds, in NICU II 43 kinds of E. coli serotype were found. In NICU I the accumulation of serotypes O6:H-, O6:H1, O19:H-, in NICU II O4:H-, O6:H1 was observed. The Klebsiella strains belonged in NICU I into 21, in NICU II into 12 phage types. Klebsiella was more frequent in NICU I than in NICU II, though the strains belonged to the same phage type in NICU II in 50.7%, but in NICU I 4 frequent and 19 rare phage types occurred. Sero- and pyocin typing was effective for typing of P. aeruginosa. The most frequent sero- and pyocin types were in NICU I:O11a,11b; in NICU II: O2a,2d,2f; 12v. The rate of antibiotic resistance in E. coli, Klebsiella, P. aeruginosa and S. aureus was nearly the same in both units, multiple resistance was more frequent in NICU I (except P. aeruginosa, it was multiple resistant in 100% in both units). In NICU I 267, in NICU II 174 infants were treated with antibiotics. The administration of penicillin derivatives was nearly similar in the two care units and the resistance among E. coli and Klebsiella strains was nearly the same too. Though, cephalosporins were used more frequently in NICU II, resistance to cephalosporins among E. coli and Klebsiella was a bit higher in NICU I. Aminoglycosides were more often used in NICU I, resistance to aminoglycosides among E. coli and Klebsiella was higher in this unit. The rate of isolation of the examined bacteria was significantly lower in the group treated with antibiotics, than in the untreated group.

Computerized complex typing of Escherichia coli strains from different clinical materials.

A multivariate analysis of 3334 Escherichia coli strains originating from different clinical materials revealed that 50.2% of isolates belonged to the most common 12 (O1, O2, O4, O6, O7, O8, O15, O18, O45, O75, O78, O83) out of 133 serogroups. Haemolysin (Hly) production, mannose resistant haemagglutinating activity for human erythrocytes (MRHA) and colicinogenicity (Col) were recorded in 30, 30 and 36%, respectively. Antigens K1 and K5 were present in 11% and 6.6%, respectively. Association were found among certain serotypes and virulence markers (O1, H-, H7, K1, MRHA, Col; O2, H-, Kl, Col; O4, H-, H5, MRHA, Hly; O6, H-, H1, MRHA, Hly; O6, K5, MRHA, Col; O7, H-, H4, K1, MRHA, Col; O18ac, H7, K1, Col; O18ac, H-, K5, MRHA, Hly; O78, H-, Col (V-type); O83, H-, K1, Col). There were associations among clinical specimens, age of patients, nosocomial group of diseases, serogroups and virulence markers, too (cerebrospinal fluid-CSF-O7, O18ac, O45, O83-K1-newborn meningitis; O78-ColV-meningitis, sepsis, inflammations diseases of premature babies; CFS-O6, MRHA, Hly-adult-meningitis, sepsis, urinary tract infection-UTI-, pneumonia, other inflammatory diseases; blood-O2, O4, O6, O18ac, ONT, K5, MRHA, Hly-sepsis, UTI, hepatic diseases; urine-O1, O2, O4, O6, O18ac, O75, virulence markers fall to differ among upper and lower UTI; faeces-O1, O4, O6, O18ac, O78, virulence markers rare). Associations were also found among animal pathogenicity tests, specimens, serogroups and virulence factors: highly virulent group strains (i.e. LD50 below 10(6)) belonged to serogroups O2, O6, O18ac, possessed antigen K1 (less frequently the presence of MRHA, Hly, K5) and originated mainly from CSF. With mouse lung toxicity test correlations of serogroups (O4, O6, O18ac), antigen K5, MRHA, Hly and specimens (blood) were also shown. However, association was found between the lack of virulence factors and phage insensitivity and also between K5 positivity and sensitivity to phages 16, 17, there were no correlations between serogroups and phage patterns. On the basis of the above-described associations one can find correlations among virulence markers, serotype, and nosological group of diseases. Animal pathogenicity tests give additional data in understanding the pathomechanism of diseases. Correlations between phage patterns and serogroups reveal certain epidemiological relatedness and also virulence of strains.

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.

[Significance of phenotypical properties in extra-intestinal E. coli infections]. / Az Escherichia coli fenotípusos tulajdonságainak jelentösége az extraintestinalis fertözésekben.

Virulence factors (serogroup, haemolysis, haemagglutination, antigens K1, K5, colicinogenicity) and their association with diseases of 3334 Escherichia coli strains isolated from different clinical specimens between 1979-1990 were analysed. Strains, that were isolated from cerebrospinal fluid of newborns under one month were characterized by certain serogroups (O7, O18, O45, O78, O83), possession of antigen K1 and production of colicin. On the basis of their LD50 they belonged to the pathogenic group (i.e. < 10 10(6)) significantly more frequently than those isolated from other materials. Strains originating from blood cultures belonged frequently to serogroup O4, O6, O18 and were haemolytic. Their pathogenicity was proved by mouse lung toxicity test. Properties of strains isolated from wound and throat swabs, urinary samples resembled to that of strains originating from blood cultures in many respects, expressing the fact that bacteria settle in different organs before sepsis is developing. Frequent occurrence of strains with antigen K1, haemolysin and haemagglutination positivity in vaginal swabs expose newborns to danger. Knowledge of virulence markers and prevention of infections are associated.

Comparison of rapid methods for detection of heat-labile (LT) and heat-stable (ST) enterotoxin in Escherichia coli.

Oxoid VET-RPLA, ST-EIA and Pharmacia Phadebact ETEC-LT enterotoxin tests were compared to find a simple but reliable method for detecting enterotoxigenic Escherichia coli (ETEC) in Hungary. In the Oxoid tests, all six reference LT- or ST-producing strains, except one ST-producer, gave positive results. Of 11 reference porcine enterotoxigenic strains, all four LT-producers gave positive reactions for LT but three of 10 ST-producers gave negative reactions for ST. Thirteen of 50 strains from culture collections of H. Steinrück (Germany) were LT+ and nine of 33 were ST+. When 31 isolates were tested simultaneously with the Oxoid and the Pharmacia LT tests, 12 strains were LT+ by the Oxoid LT test but by the Phadebact LT test only seven of these strains were LT+ and, of the remainder, three gave uncertain results and two gave negative results. Of 69 porcine strains, seven were LT+ and three ST+. Of 901 human strains isolated in Hungary, 10 were LT+ and one of 24 tested was ST+. In two cases, ETEC strains were isolated from contacts of travellers returning from Mongolia and Bangladesh. Results of comparative studies with reference strains corresponded well to those of the classical toxin detection tests. The Oxoid test was rapid, sensitive, specific and easy to perform and is recommended for use in screening ETEC isolates.

Relative surface hydrophobicity, antigen K1 and haemagglutinating activity are associated in Escherichia coli.

Hydrophobic property of 136 Escherichia coli strains was examined by salt aggregation test (SAT). Out of the tested strains 61 were SAT positive. The correlations among the surface properties characterized by SAT and other phenotypical properties, e.g. mannose resistant haemagglutinating activity (MRHA), mannose sensitive haemagglutinating activity (MSHA), presence of antigen K1 and adsorption to Al(OH)3 gel were examined. The results showed that (i) Possession of antigen K1 provides the bacterial cell a hydrophilic character and covers its relative surface hydrophobicity; (ii) Correlation exists between the relative hydrophobicity of the bacteria determined by SAT and their haemagglutinating activity. SAT values are also influenced by non haemagglutinating fimbriae and also by other non fimbrial structures; (iii) The hydrophilic surface characters are mainly expressed by the results of adsorption to Al(OH)3 gel and the hydrophobic characters rather by the SAT values.

Clonal distribution of K1 and K5 antigen possessing Escherichia coli isolates.

A significant difference was observed in the occurrence of the examined markers (Col+, ColV+, Hly+, Aer+, AbR) and in the plasmid carrier state between strains with and without K1 and K5 antigens. Plasmids of the same size were harboured by serotypes possessing K1 and K5 antigens, e.g. among O1: K1: H- strains plasmids of 60-79 Md, among O1: K1: H7, O18ac: K1: H7, O45: K1: H7 and O83: K1: H- strains plasmids of 80-95 Md were frequent. The average plasmid number was higher in K1 strains than in K5 strains. In serogroup O1 the frequency of the plasmid carrier state was associated with the O serogroup and not with the K antigen. The plasmid number in K5 of serogroups O6 and O18 was lower than in K5- strains. Plasmids of 80-95 Md were predominant among the strains derived from blood and cerebrospinal fluid, whereas these plasmids were rare among the K1 and K5 strains isolated from other sources. Plasmids of 60-79 Md were frequent among strains derived from different sources. The 30-40 Md plasmids were relatively frequent among strains isolated from urine. In contrast with literary data, O1: K1: H-, O1: K1: H7 and other frequent serotypes consisted of different clones. Different clones were found within a single serotype, too.

Cloacin tolerance and aspecific colicin insensitivity of human Escherichia coli strains.

Of 182 wild-type human, aerobactin producer Escherichia coli strains 86.3% were insensitive to cloacin. All randomly chosen 51 strains were relatively cloacin tolerant. Cloacin tolerant strains were not considerably more sensitive to hydrophobic drugs than the cloacin sensitive descendant strains. Pathogenicity of the cloacin sensitive strains was significantly lower (p less than 0.05) in intraperitoneal mice infection than that of the cloacin tolerant ones. Suggesting a new aspect of the uptake mechanism of colicins, cloacin tolerance was very frequently associated with an aspecific insensitivity to a broad spectrum of colicins.

Association of virulence markers with animal pathogenicity of Escherichia coli in different models.

Employing chicken and several strains of mice, different routes (intraperitoneal, subcutaneous) of infections and isogenic pairs of strains, association of virulence markers with animal pathogenicity was studied in Escherichia coli. Mouse virulence of avian strains was less significant than the lethality for chicks of human strains. LD50 in various animals did not differ significantly. Strains with antigen K1 were more virulent for mice than their K1- derivatives. Loss of haemolysin (Hly), mannose resistant haemagglutinating capacity or antigen K5 less markedly decreased the virulence. As opposed to other virulence factors, increased virulence of K1+ strains could also be demonstrated in mouse sepsis assay based on bacterial counts in the liver. Loss of Hly alone did not influence the persistence in the liver, however, these strains killed less mice. Aerobactin acts together with other factors, it is not per se a virulence factor. In organotropic experiments 19 strains out of 36 belonging to serotypes O7:K1:H-, O18:K1:H-, O78:H- and spontaneously agglutinable K1+ cultures, caused ophthalmitis with purulent discharge, and 4 out of 22 strains that belonged to serotype O78:H- induced uncoordinated movement of mice. Because of its special organotropic affinity to the brain and as it caused two epidemics of meningitis among newborns in Hungary, serotype O78:H- has a special pathogenic property and differs from other O78 strains that were isolated in other countries.

In vitro and in vivo (LD50) effects of human lactoferrin on bacteria.

The in vitro and in vivo effects of human lactoferrin (LF), apoLF, iron saturated LF and of different iron containing compounds (ferric chloride, ferric sodium citrate) were studied on Escherichia coli, Salmonella typhi-murium and Pseudomonas aeruginosa reference and wild-type strains with well-defined virulence markers (i.e. enterochelin, aerobactin production). LF exert in vitro antibacterial effect, and iron-free Vogel-Bonner medium proved to be suitable for its determination. The effect of intraperitoneally administered LF could not be evaluated because of its aspecificity, as any treatment (e.g. saline, Ringer solution) before bacterial challenge activated macrophages. In contrast to the in vitro results, intramuscular challenge failed to inhibit bacterial growth in vivo, as siderophores produced by bacteria were able to acquire lactoferrin-bound iron. LF treatment, like iron addition, enhanced the virulence of bacteria in mice, whereas apoLF - using iron present in the body fluids - turned to LF being unable to acquire siderophore-bound iron from bacteria. These findings do not support the literary view that LF would be useful as an antimicrobial drug.