Association of auxotypes of Neisseria gonorrhoeae and susceptibility to penicillin G, spectinomycin, tetracycline, cefaclor, cefoxitin, and moxalactam

Isolates of Neisseria gonorrhoeae from 304 patients attending a venereal disease clinic were examined by a plate dilution method for their susceptibility to six antibiotics: penicillin G, spectinomycin, tetracycline, cefaclor, cefoxitin, and moxalactam. The isolates were also characterized by gonococcal auxotyping. The most frequent auxotypes were Nonrequiring, 58%; Pro-, 14%; Pro- Arg (Orn*) Ura-, 14%; Arg- Hyx- Ura-, 6%; and a miscellaneous group consisting of 8% of the isolates. If the entire group of isolates were examined, moxalactam was the most active of the antibiotics; 94% of the isolates were inhibited by less than or equal to 0.25 microgram/ml. The Pro- Arg (Orn*) Ura- isolates were relatively resistant to penicillin G and cefoxitin. The Arg- Hyx- Ura- group of isolates was the most susceptible of the auxotypes to all of the antibiotics except spectinomycin. The uncommon auxotype Arg (Orn*) Ura- has a requirement for arginine that is satisfied by citrulline but not by ornithine. The results of the present study indicate that the nutritional requirements of gonococci may be associated with their response to certain antibiotics.     

Susceptibility of different auxotypes of Neisseria gonorrhoeae to norfloxacin, cefotaxime, cefuroxime, and erythromycin

Isolates of Neisseria gonorrhoeae from 150 patients attending a venereal disease clinic were examined for their susceptibility to four antibiotics (norfloxacin, cefotaxime, cefuroxime, and erythromycin) by a plate-dilution method. The isolates were also characterized by gonococcal auxotyping. The most frequent auxotypes were nonrequiring, 50%; Pro-, 16.7%; Pro-, Arg-, (Orn*), Ura-, 10.7%; Arg-, Hyx-, Ura-, 12.7%; and a miscellaneous group consisting of 10% of the isolates. Norfloxacin was the most active of the antibiotics; all of the isolates were inhibited by less than or equal to 0.06 microgram/ml. The Arg-, Hyx-, Ura- group of isolates was the most susceptible of the auxotypes to antibiotics. In the present study N. gonorrhoeae of the Arg-, Hyx-, Ura- auxotype fell into two groups with respect to erythromycin, one highly susceptible and one less susceptible. The results of the present study provide further evidence that the nutritional requirements of gonococci are associated with their response to certain antibiotics.     

Chemotaxis of polymorphonuclear leukocytes from donors of different races toward Neisseria gonorrhoeae of three different auxotypes

Urethral gonorrhea is typified by the presence of large numbers of polymorphonuclear leukocytes, which are presumably attracted to the urethra by an as yet uncharacterized chemotaxin. Culture supernatants from three different auxotypes of Neisseria gonorrhoeae, one requiring arginine, hypoxanthine, and uracil (Arg-,Hyx-,Ura-), one requiring proline, arginine (not satisfied by ornithine), and uracil (Pro-,Arg-[Orn*],Ura-), and one requiring proline (Pro-), were tested for their chemotactic activity against leukocytes from men of two racial groups, white and black. These auxotypes were chosen because the Pro- auxotype is isolated more frequently from black patients, and the Arg-,Hyx-,Ura- and the Pro-,Arg-(Orn*),Ura- auxotypes are isolated more frequently from white patients. Chemotaxis was tested in blind-well chambers in the absence of complement; membranes with a pore size of 3 microns were used. The culture supernatants of the Pro- auxotype were the most chemotactic, while those of the Arg-,Hyx-,Ura- were the least chemotactic when analyzed by a three-way analysis of variance: F = 22.83, df = 2.6, P less than .001. There were no differences in migration with respect to the race of the donor. Gonococcal culture supernatants did not require complement for chemotaxis.     

Auxotypes of Neisseria gonorrhoeae isolated from heterosexual men, homosexual men, and heterosexual women

During 1979-1984 we auxotyped 1822 strains of Neisseria gonorrhoeae and recorded the site of isolation and sexual orientation of the patients. Auxotypes were determined by the growth requirements of strains for proline (Pro-), uracil (Ura-), hypoxanthine (Hyx-), citrulline (Cit-), or citrulline replaceable by ornithine (Orn-). Of all isolates from homosexual men, 96% belonged to three auxotypes: nonrequiring (NR), Pro-, or Orn-, and only 1.5% belonged to the Pro-, Cit-, Ura- and Orn-, Ura-, Hyx- auxotypes. Of the isolates from women, 49.9% belonged to these latter two auxotypes. Of the strains isolated from male homosexuals, 19.5% were resistant to 1.0 microgram of erythromycin/ml, whereas only 9.6% of strains from other men and 2.6% of strains from women were resistant to this concentration. We suggest that strains of N. gonorrhoeae infecting homosexual men tend to be less demanding in their nutritional requirements and more resistant to erythromycin than strains infecting heterosexual men and women.     

Characteristics of Neisseria gonorrhoeae strains isolated on selective and nonselective media

The possible inhibition of Neisseria gonorrhoeae on modified Thayer-Martin (VCNT) medium was investigated by inoculation of multiple media with specimens from 3,490 patients. N. gonorrhoeae was recovered from 461 patients, and in 24 cases (5.2%) it was isolated on drug-free medium only; 18% of the recoveries were on VCNT medium only. The minimal inhibitory concentration (MIC) of benzylpenicillin was determined for 411 of the strains, and 175 were examined for responses to 12 other antibiotics and for auxotype. Of the 24 strains isolated on drug-free medium only, one was inhibited by trimethoprim at a concentration of 2.0 micrograms/ml and four others had MICs of vancomycin of less than or equal to 2.0 micrograms/ml. The remainder were resistant to vancomycin, trimethoprim, and colistin at the concentrations present in VCNT medium. Unexpectedly, four strains isolated on both VCNT and drug-free medium had MICs of vancomycin of less than or equal to 3.0 micrograms/ml and were defined as hypersusceptible. Genetic tests showed that gonococci resistant to less than or equal to 0.5 microgram of vancomycin/ml differed genotypically from those resistant to 1.0 microgram/ml. The eight strains hypersusceptible to vancomycin were highly susceptible to various other antibiotics. Their nutritional requirements included hypoxanthine (Hyx-) and uracil (Ura-), and all but one also required arginine (Arg-), which for two strains could not be replaced by ornithine (Arg0-). Pro-,Arg0-,Ura- (5.7%) and Arg0-,Ura- (1.1%) auxotypes were found at this time but not in earlier studies of gonococci isolated in the same clinic.     

Bactericidal properties of urine for Neisseria gonorrhoeae

Some human urine is bactericidal for the F-62 strain of Neisseria gonorrhoeae. Gonococci of three auxotypes (Pro-; Arg-, Hyx-, Ura-; and Pro-, Arg-. (Orn*), Ura-) were tested by in-vitro exposure to 31 samples of urine from 14 men. Nineteen of the urine specimens were bactericidal, and 12 were not. Except for one sample, all cidal urines came from five men. Cidal activity was associated with acidic, concentrated urines; it was unaffected by exposure to lowered pH, pronase, heat or cold, and was dialyzable with use of a dialysis membrane with a cut-off molecular weight of 1000. Neutralization of the acid urines removed the antigonococcal activity. Noncidal acid urines became cidal urines when concentrated by lyophilization. Zinc, lysozyme, fluoride ions, and fatty acids are substances that have antibacterial activity and are also present in urine. These substances were examined for antigonococcal activity. Neither zinc salts, fluoride ions, lysozyme, nor fatty acids in concentrations exceeding those found in urine were bactericidal for the gonococci. These results show that sufficiently concentrated, acidic urines kill gonococci by an unknown mechanism.     

Neisseria gonorrhoeae: subdivision of auxogroups by genetic transformation.

Genetic transformation was used in an attempt to subdivide the most prevalent auxotypes of Neisseria gonorrhoeae in local isolates. The large proline requiring (Pro-) group could be divided into two genetic types, as could the less common arginine requiring (Arg-) group. The large arginine, hypoxanthine, and uracil requiring (Arg- Hyp- Ura-) group could not be subdivided by this method. The genetic relation between these and other auxotypes was investigated.     

Serogrouping of Neisseria gonorrhoeae: correlation of serogroup with disseminated gonococcal infection

Gonococci can be divided into serogroups WI, WII, and WIII by coagglutination. To assess the clinical correlation of serogroups of gonococci, we studied isolates of gonococci from patients with disseminated and uncomplicated gonococcal infections in three cities in the United States. Strains of Neisseria gonorrhoeae belonging to serogroup WI represented 85 (84%) of 101 isolates from patients with disseminated gonococcal infection (DGI) and 68 (40%) of 168 isolates from patients with uncomplicated gonorrhea (P less than .001). The auxotype Arg-, Hyx-, Ura- (AHU) represented 62 (61%) of 101 isolates from DGI and 54 (32%) of 168 isolates from uncomplicated infection (p less than .001); all AHU isolates were serogroup WI. Among other auxotypes, WI strains represented 25 (64%) of 39 isolates from patients with DGI and 14 (12%) of 114 from uncomplicated infections (P less than .001). In Atlanta only, 13 (34%) of 38 isolates from DGI were AHU, but 31 (82%) were serogroup WI (P less than .001). Thus gonococci of serogroup WI are associated with DGI in these cities of the United States, and this correlation appears to be independent of auxotype. Serogroup WI is correlated with serum resistance.     

Auxotypes and antimicrobial susceptibilities of Neisseria gonorrhoeae in black and white patients.

Isolates of Neisseria gonorrhoeae from 100 white and 113 black patients attending a venereal disease clinic were examined for their susceptibility to ampicillin, penicillin, tetracycline, and spectinomycin. The isolates were also characterised by gonococcal auxotyping. Gonococcal isolates from black patients were more resistant to the antibiotics than those from white patients, since the former were infected with the more antibiotic-resistant auxotypes (Pro, Zero, and Arg) whereas the latter were infected with the more antibiotic-susceptible auxotypes (AHU and others). These data indicate a preferential infection by sub-populations of Neisseria gonorrhoeae in two racial groups.     

Auxotypes and antimicrobial susceptibilities of Neisseria gonorrhoeae in black and white patients

Isolates of Neisseria gonorrhoeae from 100 white and 113 black patients attending a venereal disease clinic were examined for their susceptibility to ampicillin, penicillin, tetracycline, and spectinomycin. The isolates were also characterised by gonococcal auxotyping. Gonococcal isolates from black patients were more resistant to the antibiotics than those from white patients, since the former were infected with the more antibiotic-resistant auxotypes (Pro, Zero, and Arg) whereas the latter were infected with the more antibiotic-susceptible auxotypes (AHU and others). These data indicate a preferential infection by sub-populations of Neisseria gonorrhoeae in two racial groups.     

Antibiotic susceptibilities, serotypes and auxotypes of Neisseria gonorrhoeae isolated in New Zealand.

OBJECTIVE--The aim of the study was to determine the distribution of auxotypes and serotypes and the prevalence of antibiotic resistance among New Zealand isolates of Neisseria gonorrhoeae. MATERIALS AND METHODS--A total of 486 gonococci isolated in 1988 were auxotyped, serotyped, and tested for susceptibilities to ten antibiotics. RESULTS--The gonococci were susceptible to all the antibiotics tested except penicillin and tetracycline. Eleven (2.2%) produced beta-lactamase, one (0.2%) showed chromosomal penicillin resistance, and 18 (3.7%) were resistant to a low-level of tetracycline. Most of the gonococci belonged to six auxotypes. The three predominant auxotypes were arginine-requiring (Arg-), non-requiring (NR), and arginine, hypoxanthine, uracil-requiring (AHU-). The majority of the isolates belonged to serogroup IB and to six serovars. The most prevalent serovars were IB-3 and IB-1. There was an association between penicillin susceptibility and auxotype or serovar among non-penicillinase producing N. gonorrhoeae (PPNG) isolates. CONCLUSIONS--Antibiotic resistance, including penicillin resistance, remains uncommon among gonococci in New Zealand. Baselines have been established for future epidemiological studies using both auxotyping and serotyping.     

Association between auxotypes, serogroups, and antibiotic susceptibilities of Neisseria gonorrhoeae isolated from women in Mumbai (formerly Bombay), India.

OBJECTIVES: Gonococcal isolates were differentiated based on susceptibility pattern, penicillinase production (PPNG or non-PPNG), serogroup, auxotype, protein, and plasmid profile. The association between serogroup and auxotype and PPNG was determined. STUDY DESIGN: Women attending tertiary level health centers and the sexually transmitted disease (STD) clinic in Mumbai, India, were screened for Neisseria gonorrhoeae. Minimal inhibitory concentration testing was performed according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Auxotypes, serogroups, protein profile, and plasmid content were also studied. RESULTS: Of the 33 isolates, 16 (48.5%) were resistant to penicillin, and 28 (84.8%) showed a chromosomally mediated resistance to tetracycline. Five (15.2%) isolates showed resistance to ciprofloxacin, whereas 12 (36.4%) showed a reduced susceptibility. Twenty-seven (81.8%) isolates belonged to the WI serogroup, and 15 (46.7%) were penicillinase producers (PPNG). Seventeen (51.5%) isolates were of the nonrequiring auxotype, whereas seven (21.2%) were proline requiring. Fifteen (55.6%) of the isolates belonged to the nonrequiring-WI auxotype/serogroup (A/S) class. Ten of the PPNG isolates possessed the 4.4 MDa plasmid, whereas four had the 3.2 MDa plasmid. Increases in the molecular weight of the major outer membrane protein were observed. CONCLUSION: A high prevalence of chromosomal resistance to penicillin and tetracycline was observed. The 4.4 MDa plasmid was the most prevalent among the PPNG isolates. We observed ciprofloxacin resistance, which has not been reported in previous studies in India. The nonrequiring auxotype was the most prevalent, followed by the proline requiring auxotype. WI serogroup was the most commonly observed among the isolates studied. The nonrequiring/WI A/S class was the most prevalent among the PPNG.     

Nutritional requirements and penicillin susceptibilities of gonococci from pharyngeal and anogenital sites.

Neisseria gonorrhoeae was isolated from 5.9% of oropharyngeal specimens obtained from patients attending a clinic for sexually transmitted diseases. Oropharyngeal isolates from 69 patients and anogenital isolated from 97 other patients attending the same clinic were compared. Many of the gonococci could be differentiated by the compounds required for growth in chemically defined media or by differences in the minimum inhibitory concentration (MIC) of penicillin G. Strains with requirements for either proline (Pro-) or arginine (Arg-) or for none of the compounds that are used for differentiation (zero phenotype) were more common in the oropharynx (91.3% of patients) than in anogenital sites (73.2% of patients). On the other hand, gonococci with multiple requirements that include arginine, hypoxanthine, and uracil (AHU strains) were present in oropharyngeal specimens from only three patients (4.4%), but were isolated from anogenital specimens from 18 patients (18.6%). A high susceptibility to penicillin characterised the AHU strains from all sites, as others have reported. The penicillin MIC ranged from 0.003-0.72 microgram/ml for strains with Pro-, Arg-, and zero phenotypes. However, a penicillin MIC greater than or equal to 0.42 microgram/ml was found for 17.6% of oropharyngeal isolates of these types, but for only 4.1% of Pro-, Arg-, and zero isolates from anogenital sites. None of these moderately resistant strains produced beta-lactamase. Our findings indicate that gonococci differ in their ability to colonise the oropharynx successfully.     

Nutritional requirements and penicillin susceptibilities of gonococci from pharyngeal and anogenital sites.

Neisseria gonorrhoeae was isolated from 5.9% of oropharyngeal specimens obtained from patients attending a clinic for sexually transmitted diseases. Oropharyngeal isolates from 69 patients and anogenital isolated from 97 other patients attending the same clinic were compared. Many of the gonococci could be differentiated by the compounds required for growth in chemically defined media or by differences in the minimum inhibitory concentration (MIC) of penicillin G. Strains with requirements for either proline (Pro-) or arginine (Arg-) or for none of the compounds that are used for differentiation (zero phenotype) were more common in the oropharynx (91.3% of patients) than in anogenital sites (73.2% of patients). On the other hand, gonococci with multiple requirements that include arginine, hypoxanthine, and uracil (AHU strains) were present in oropharyngeal specimens from only three patients (4.4%), but were isolated from anogenital specimens from 18 patients (18.6%). A high susceptibility to penicillin characterised the AHU strains from all sites, as others have reported. The penicillin MIC ranged from 0.003-0.72 microgram/ml for strains with Pro-, Arg-, and zero phenotypes. However, a penicillin MIC greater than or equal to 0.42 microgram/ml was found for 17.6% of oropharyngeal isolates of these types, but for only 4.1% of Pro-, Arg-, and zero isolates from anogenital sites. None of these moderately resistant strains produced beta-lactamase. Our findings indicate that gonococci differ in their ability to colonise the oropharynx successfully.     

Antibiotic susceptibility in relation to serogroup and auxotype of Neisseria gonorrhoeae isolates from Gabon

We tested 75 beta-lactamase-positive (penicillinase-producing Neisseria gonorrhoeae; PPNG) and 147 beta-lactamase negative (non-PPNG) strains of N. gonorrhoeae obtained during a two-year period in Franceville, a semirural community in southeastern Gabon, for susceptibility to antimicrobial agents, auxotype, and serotype. The most common auxotypes were the nonrequiring and proline-requiring strains. Of the non-PPNG strains tested, 37% belonged to serogroup WI, 48% to serogroup WII, and 15% could not be grouped with commercially available antisera. Except for four strains that belonged to serogroup WII, the PPNG strains were classified as serogroup WI or ungroupable. We determined the concentrations at which 90% of strains were inhibited (MIC90) for spectinomycin (32 mg/liter for both PPNG and non-PPNG), kanamycin (32 mg/liter for both PPNG and non-PPNG), tetracycline (2 mg/liter for non-PPNG, 1 mg/liter for PPNG), cefotaxime (0.03 mg/liter for non-PPNG, 0.008 mg/liter for PPNG), and penicillin (1 mg/liter for non-PPNG, greater than 32 mg/liter for PPNG). A MIC of greater than or equal to 2 mg of tetracycline/liter was noted for 17% of the strains. Strains belonging to serogroup WII were more resistant to tetracycline and cefotaxime than other strains. Proline-requiring strains were less susceptible to tetracycline, cefotaxime, and spectinomycin than the nonrequiring strains. The decreased sensitivity to penicillin (non-PPNG strains) and tetracycline was determined by proline-requiring strains belonging to serogroup WII.     

淋球菌营养型与耐药性及质粒的关系

目的 为了解淋球菌的营养型、耐药性及质粒之间的关系.方法 对重庆市收集的83株淋球菌进行营养型、抗生素敏感性和质粒检测.结果 83株菌共分为8个营养型,以Pro-型(51-8%)和Proto型(18-1%)为主.利用琼脂稀释法检测菌株对青霉素、四环素和壮观霉素的敏感性.青霉素耐药株占15.6%,四环素耐药株占70%,壮观霉素耐药株占1-2%.通过快速碱裂解法对其中的44株菌进行质粒检测,70%菌株含24-5Md质粒,80%含2.6Md质粒,66%同时含24-5Md和2.6Md质粒,16%未查到质粒.并发现Pro^-型菌株中青霉素耐药株高于其它营养型(P<0.05),且含24-5Md质粒菌也显著高于其它营养型(P<0.05).含24-5Md+2.6Md质粒菌株中四环素耐药株更常见(P<0.05).检测到1株产青霉素酶淋球菌(PPNG),经质粒消除试验证实其耐药性由4-4Md质粒介导.结论 本研究不仅表明了重庆地区淋球菌营养型、耐药性及质粒的分布状况,而且揭示了它们之间存在一定关系.这将对该地区淋球菌的流行病学调查和淋病防治提供帮助.     

Auxotypes and antibiotic susceptibilities of Neisseria gonorrhoeae from women with acute salpingitis. Comparison with gonococci causing uncomplicated genital tract infections in women

An investigation of the MICs of various antibiotics and the nutritional requirements (auxotypes) of Neisseria gonorrhoeae recovered from the cervix, fallopian tubes, and peritoneal cavity of women with acute salpingitis was done. These MICs and auxotypes were compared to those of gonococci isolated from the cervix of women with uncomplicated or asymptomatic genital tract gonorrhea. The MICs of minocycline, penicillin G, ampicillin, cefoxitin, and cefaclor for gonococci isolated from women with acute salpingitis were significantly higher. Significant differences in auxotype patterns were identified between isolates from salpingitis cases and uncomplicated cases. The prototrophic form was the most common (75%) among salpingitis strains. No strains requiring arginine, hypoxanthine, and uracil were identified among the salpingitis isolates. In contrast, strains that required these nutrients were the most frequent auxotype among isolated from women with uncomplicated genital tract gonorrhea. Discriminant analysis revealed that the MIC of minocycline and the auxotype were the most powerful discriminators between groups of patients with different manifestations of gonorrhea.     

Antimicrobial susceptibility, auxotype and plasmid content of Neisseria gonorrhoeae in northern Tanzania: emergence of high level plasmid mediated tetracycline resistance.

OBJECTIVE--To study the antimicrobial susceptibility, plasmid content, auxotype and serogroup of strains of Neisseria gonorrhoeae isolated from an urban population of STD clinic attenders in Northern Tanzania. METHODS--The minimum inhibitory concentrations of nine common antimicrobial agents were measured by the agar dilution method against 130 strains of Neisseria gonorrhoeae isolated in a free government STD clinic in Mwanza town. The auxotype, plasmid content and serogroup of these strains were also determined by conventional techniques. RESULTS--65 strains (50%) were penicillinase producers (PPNG), and 34 (26%) exhibited chromosomally mediated resistance to penicillin. Seven (5%) were sensitive to tetracycline; 78 (60%) showed intermediate levels of resistance, and 45 (35%) had high level plasmid mediated resistance (TRNG), all of which carried a 25.2 MDa plasmid. 79 strains (61%) showed decreased sensitivity to trimethoprim-sulphamethoxazole, and five (4%) were resistant to this agent. All isolates were fully sensitive to spectinomycin, azithromycin, cefotaxime, cefuroxime, norfloxacin and ciprofloxacin. One hundred and one strains (78%) were of type W11/111, 22 type W1, and seven cross reacting strains. The W1 strains were significantly more likely to be carrying plasmid mediated resistance to both penicillin and tetracycline. Six different auxotypes were present, the major type requiring proline. Plasmid profiles showed the presence of both the 3.2 MDa and the 4.4 MDa beta-lactamase encoding plasmids. CONCLUSION--a high proportion of gonococcal isolates remain resistant to penicillin in this region, and most isolates are now also resistant to tetracycline, with the emergence of plasmic mediated tetracycline resistance. Trimethoprim-sulphonamide sensitivity is also decreasing. The population of strains is heterogeneous, and both African and Asian beta-lactamase encoding plasmids are present.     

Antibiotic resistance, plasmid profile, auxotypes and serovars of Neisseria gonorrhoeae strains isolated in Sevilla (Spain).

The antibiotics resistance pattern, the plasmid profile, the auxotypes and serotypes of 116 Neisseria gonorrhoeae clinical isolates obtained in one year were examined. The incidence of penicillinase producing (PPNG) strains was 12% (14 strains). The most frequent plasmid pattern was the combination of 4.5, 2.6 and 24.5 MDa plasmids. The conjugative plasmid of 24.5 MDa showed a high prevalence (32% of the total strains), and almost all the PPNG strains harboured this plasmid. The strains with the 4.5 MDa plasmid belonged to the auxotypes Pro-, Zero and Pro-Hyx-Ura-, whereas that with the 3.2 MDa plasmid was of auxotype Pro-Hyx-His-. The serotypes Aedih/Arst (WI serogroup) and Bak/Bropt, Back/Bropyt and Bak/Bropyt (WII/III serogroup) were predominant.     

Auxotypes and sensitivity to 6 antibiotics of strains of Neisseria gonorrhoeae isolated at Strasbourg in 1977-1978 (author's transl)]

We have studied 386 strains of Neisseria gonorrhoeae isolated in our laboratory from July 1977 to October 1978, with the Catlin's auxotyping method. The distribution of the four main auxotypes, i. e. (+), (PRO-), (ARG-), (ARG-, HYX-, URA-), is different from those reported by others (in U. S. A. and Sweden). In Strasbourg, the auxotype (+) is prevalent. We have noticed some differences among classes of population. For example (ARG-) and (ARG-, HYX-, URA-) are scarcely isolated from prostitutes and we have found less (ARG-) strains in women than in men. The determination of the minimum inhibitory concentration of 6 antibiotics has shown there was no differences between the auxotypes except for the (ARG-, HYX-, URA-) strains which are more susceptible to all the antibiotics, but spectinomycin. Since August 1978, we have also noticed an increasing of the resistance which is mainly due to (PRO-) strains.