Neisseria lactamica and Neisseria polysaccharea as Possible ...

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CHEMOTHERAPY, Nov. 1990, p. 2269-2272

Vol. 34, No. 11

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Neisseria lactamica and Neisseria polysaccharea as Possible Sources of Meningococcal 3-Lactam Resistance by Genetic Transformation JUAN A. SAEZ NIETO,l* RODOLFO LUJAN,1 JOAQUIN V. MARTINEZ-SUAREZ,' SONSOLES BERRON,1 JULIO A. VAZQUEZ,' MIGUEL VINAS,2 AND JOSE CAMPOS3

Laboratorio de Referencia de Meningococos, Servicio de Bacteriologia, Centro Nacional de Microbiologia, Virologia e Immunologia Sanitarias, 28220 Majadahonda, Madrid,' and Departamento de Microbiologia, Facultad de Farmacia, Universidad de Barcelona, Barcelona,2 and Servicio de Microbiologia, Hospital de San Juan de Dios, 08032 Barcelona,3 Spain Received 28 March 1990/Accepted 17 August 1990

We studied the susceptibilities of relatively penicillin G-resistant and -susceptible strains of Neisseria meningitidis, as well as Neisseria lactamica and Neisseria polysaccharea, to penicillin, ampicillin, and several cephalosporins. The MICs of penicillin, ampicillin, cephalothin, and cefuroxime for moderately resistant meningococci have increased two- to sixfold in relation to MICs for susceptible strains. For these strains of meningococci, N. lactamica, and N. polysaccharea, penicillin, ampicillin, cephalothin, and cefuroxime MICs for 50 and 90% of strains were similar. By genetic transformation of a penicillin-susceptible strain of N. meningitidis to low-level penicillin resistance with DNA from penicillin-resistant strains of N. meningitidis, N. lactamica, N. polysaccharea, and N. gonorrhoeae, isogenic strains with the same pattern of resistance to beta-lactams were obtained, suggesting that these commensal Neisseria spp. could be the source of meningococcal resistance genes.

The National Reference Laboratory for Meningococci has carried out a microbiological surveillance in Spain (1978 to 1989) of 4,448 meningococcal strains isolated from blood or spinal fluid of patients (15). After describing a meningococcal isolate in 1985 with an MIC of penicillin of 0.2 ,ug/ml and nine isolates in 1986 with MICs of penicillin between 0.2 and 0.4 p,g/ml (0.4 and 5.3%, respectively, of strains isolated from patients and studied over the years), the percentages of moderately resistant strains (Penr) increased 7 and 20% during successive years (1987 to 1989) (12, 14; J. A. Saez-Nieto, submitted for publication). In recent years clinical isolates of Neisseria meningitidis with increased levels of penicillin resistance have been described in other countries (3, 18) and some strains have been described with MICs of penicillin of >1 ,ug/ml (11, 18). In this study, we have compared the susceptibilities of the penicillin-resistant strains with those of susceptible strains as regards susceptibilities of the strains to several alternative antibiotics for treatment and prophylaxis of meningococcal infections. We have also studied the susceptibilities to the same antimicrobial agents of N. lactamica and N. polysaccharea, which are species very similar to the meningococci in habitat, genetic distance, and antigenic structure. We suggest the possibility of penicillin resistance transmission into meningococci by genetic transformation from commensal neisseriae. We have carried out transformation experiments with DNAs from these species, obtaining moderately resistant transformants of similar phenotypes in all

ampicillin, Penr Ampr; 30 moderately resistant to ampicillin but susceptible to penicillin, Pens Ampr; and 50 susceptible strains, Pens Amps). For comparative purposes, 30 N. lactamica and 30 N. polysaccharea strains isolated from nasopharynges of children were used; these strains have been described previously (2, 13). For transformation experiments the meningococcus prototype strain H44/76 (B15 P1.16) with an MIC of penicillin of 0.025 ,ug/ml was used. The DNA donors used were five strains of N. meningitidis and one each of N. lactamica, N. polysaccharea, and N. gonorrhoeae (see Table 2). The maintenance, growth conditions, and differential methods for Neisseria species have been previously described (2, 9). All the strains were stored in skim milk (Difco Laboratories) at -70°C. The test conditions used for susceptibility testing of antibiotics were those previously described for meningococci (14). Similar methods were used for the N. lactamica and N. polysaccharea strains. Briefly, Mueller-Hinton agar (Difco) supplemented with 5% sheep blood was used for growth and susceptibility studies. Plates were incubated for 18 h at 36°C in air in a humid atmosphere. The strains were suspended in phosphate-buffered saline, pH 6.9, with density adjusted to a 0.5 McFarland standard. Each of these suspensions was adjusted by dilution to achieve an inoculum of 105 CFU per spot by using an automatic multi-inoculator (Microtiter AM80). Chromosomal DNA was prepared from sodium dodecyl sulfate-lysed cells by a previously described method (6) with phenol-chloroform extraction and ethanol precipitation for final purification. N. meningitidis H44/76 was transformed by a method similar to that described for the gonococci (1), with some modifications. The recipient strain was harvested after growth by being scraped from an 18-h culture on GC base agar (Difco) and suspended to provide a density of 5 x 107

cases.

A total of 210 strains of N. meningitidis isolated between 1985 and 1989 from patients from Spanish provinces were studied (130 moderately resistant to both penicillin and

*

Corresponding author. 2269

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NOTES TABLE 1. Comparative activities of beta-lactam antibiotics Organism (no. of isolates)

MIC 4LgIml)a Antibiotic

Range

50%

90%

N. meningitidis, Penr Ampr (130)

Penicillin Ampicillin Cephalothin Cefoxitin Cefuroxime Cefotaxime Ceftriaxone

0.1-0.8 0.1-0.8 0.4-3.2 0.1-0.4 0.1-1.6 0.003-0.05 s0.0003-0.003

0.2 0.35 1.3 0.2 0.25 0.004 0.0005

0.35 0.7 2.4 0.3 0.6 0.008 0.001

N. meningitidis, Pens Ampr (30)


0.012-0.05 0.1-0.4 1.6-3.2 0.1-0.4 0.1-0.4 0.003-0.006 s0.0003-0.0015

0.025 0.2 1.6 0.2 0.2 0.003 0.0006

0.05 0.4 2.4 0.3 0.35 0.005 0.001

N. meningitidis, Pens Amps (50)


s0.012-0.05 s0.012-0.05 0.2-0.8 0.1-0.4 0.012-0.1 0.0015-0.006 s0.0003-0.0015

0.025 0.03

0.05

0.2 0.15 0.02 0.002 0.0005

0.05 0.4 0.2 0.05 0.003 0.0005

N. lactamica (30)


0.1-0.8 0.2-0.8 1.6-3.2 0.1-0.4 0.1-0.8 s0.003-0.025 s0.0003-0.0015

0.2 0.4 1.8 0.15 0.3 0.006 0.0007

0.4 0.7 2.9 0.2 0.6 0.02 0.0015

N. polysaccharea (30)


0.05-0.8 0.2-1.6 0.4-3.2 0.1-0.2 0.05-0.8 s0.003-0.025

0.25 0.45 1.5 0.15 0.3 0.004 0.0004

0.3 0.8 2.8 0.2 0.5 0.008 0.001

s0.0003-0.0015

a 50% and 90%, MIC for 50 and 90%o of isolates, respectively.

CFU/ml in 0.9 ml of GCB broth containing 5 mM CaCl2. Transforming DNA (0.1 ml) was added to a saturating concentration (>1 ,ug/ml) and was incubated together with the cells at 37°C for 30 min. Controls were processed identically except for the transforming DNA. The cells were incubated for phenotypic expression of antibiotic resistance for 6 h on GCB agar plates before addition of a soft agar overlay containing penicillin at a concentration of 0.05 ,ug/ml. The results obtained with beta-lactam antibiotics are shown in Table 1. The MICs of penicillin against Penr strains ranged from 0.1 to 0.8 ,ug/ml. All the N. lactamica isolates were Penr; only two strains of N. polysaccharea had MICs of penicillin of 0.05 ,ug/ml, but these were moderately resistant to ampicillin, as were the rest of N. polysaccharea strains. No significative differences were found in the activities of cefoxitin, cefotaxime, and ceftriaxone against these bacterial groups. Table 2 shows the origins and properties of moderately penicillin-resistant transformants from the H44/76 meningococcal strain. Transformants obtained with DNA from Penr meningococci at the first transformation level were only four times more resistant to penicillin than recipient strains.

Similar results were obtained when DNAs from N. lactamica and N. polysaccharea were used (Table 2). To compare this low-level penicillin resistance of the transformants with that of gonococci, the PenA mutation from N. gonorrhoeae FA102 (16) was transformed into the H44/76 meningococcal strain. The level of penicillin resistance attained by these transformants was 0.2 ,ug/ml, representing an eightfold increase over the MIC of penicillin for the recipient meningococcus, an increase similar to that observed when gonococcal PenA was transformed into susceptible gonococci. A similar pattern of resistance to other beta-lactam antibiotics was found in transformants of all four species. As in the original strains, transformants showed increased MICs of cephalothin and cefuroxime but not of cefoxitin, cefotaxime, and ceftriaxone (Table 2). Meningococci that are relatively resistant to penicillin have been increasingly isolated in Spain (0.4% in 1985 to 20% in 1989). The emergence of such strains during this period suggests a change in behavior of N. meningitidis in relation to its susceptibility to penicillin and other betalactams. Recently, 3-lactamase-producing meningococcal strains in Spain and Southern Africa (3, 7) and penicillin-resistant

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TABLE 2. MICs of transformants of susceptible meningococcal strain H44/76 with DNAs of Penr meningococcal strains and other Neisseria species MIC (,ug/ml)

Straina

Penicillin

Ampicillin

Cephalothin

Cefuroxime

Cefoxitin

Cefotaxime

Ceftriaxone

H44/76 6037 Tll (6037) 5959 T21 (5959) 7116 T711 (7166) 7121 T811 (7121) 7188 T911 (7188) N. lactamica 6546 T61 (6546)

0.025 0.4 0.1 0.2 0.1 0.8 0.2 0.4 0.1 0.4 0.1 0.4 0.1

0.025 0.8 0.4 0.4 0.4 0.8 0.4 0.8 0.4 0.8 0.4 0.8 0.2

0.4 3.2 1.6 3.2 1.6 1.6 1.6 1.6 0.8 3.2 1.6 3.2 0.8

0.025 0.8 0.2 0.8 0.4 0.4 0.4 0.2 0.1 0.8 0.4 0.4 0.1

0.2 0.4 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

0.003 0.012 0.012 0.025 0.025 0.006 0.006 0.006 0.006 0.012 0.012 0.003 0.003

0.0015 0.003 0.003 0.003 0.0015 0.0015 0.0015 0.003 0.0015 0.003 0.0015 0.0015 0.0015

N. polysaccharea 5818 T511 (5818)

0.4 0.1

0.8 0.4

3.2 0.8

0.2 0.2

0.2 0.2

0.003 0.003

0.0015 0.0015

N. gonorrhoeae FA102 T31 (FA102)

0.05 0.2

0.1 0.2

0.8 0.8

0.2 0.1

0.2 0.2

0.003 0.003

0.0015 0.0015

N. meningitidis

a H44/76 is the susceptible recipient strain; strains 6037, 5959, 7166, 7121, and 7188 are the meningococcal Penr DNA donors. Strains Tll, T21, T711, T811, T911, T61, T511, and T31 are transformants of H44/76 meningococcal strain with DNA of the Penr Neisseria species in parentheses.

strains of non-,B-lactamase producers with MICs of penicillin of >1 ,ug/ml have been described (11, 18). The Penr resistance mechanism in ,3-lactamase-negative meningococci has been established and consists of a reduced affinity of penicillin-binding protein 3 (PBP 3) of meningococci (8); however, as proposed in a recent article by Spratt et al. (17), the homology of PBP 3 of meningococci and PBP 2 of gonococci suggests that PBP 2 of gonococci should be referred to as PBP 2 of meningococci. In our study, we have found that the Penr Ampr and Pen' Ampr strains showed similar MICs for other beta-lactams. An increase in the MICs of narrow- and expanded-spectrum cephalosporins (cephalothin and cefuroxime) was found, suggesting a similar resistance mechanism. No differences were observed for broad-spectrum cephalosporins (cefotaxime and ceftriaxone) and cefoxitin. This increase of MICs of cephalothin and cefuorxime has been recently described by other authors (10) and detected in earlier works by disk susceptibility testing (4, 14). Recently Spratt et al. have analyzed the genes coding for altered PBP 2 of N. meningitidis, N. flasvescens, and N. polysaccharea compared with the gonococcal PBP 2 gene. They found the genes to be very similar to the Penr gene in gonococci and meningococci (17). N. lactamica and N. polysaccharea could also have similar altered PBP 2 genes (R. Lujan et al., submitted for publication). We were able to transform a type strain of N. meningitidis to low-level penicillin resistance with DNA from different Neisseria species, obtaining similar patterns of resistance to beta-lactam antibiotics. This may suggest the existence of similar resistance mechanism in both N. meningitidis and other commensal neisseria. For these reasons and because of the special characteristics of these commensal neisseriae with respect to meningococcus (higher percentage of carriers, resistance, and the same habitat), the commensal neisseriae could be a reservoir of beta-lactam antibiotic resistance for the meningococcus. Finally, we think that the continuous surveillance of the Penr strains of meningococci in other countries could be very

important for detecting the emergence of strains with MICs of penicillin greater than 1 ,ug/ml in the near future, which could be a serious problem in the treatment of meningococcal infections. However, a good alternative for therapy is the broad-spectrum cephalosporins, because the MIC ranges obtained for Penr and Pens meningococci are very low and far from levels obtained in cerebrospinal fluid by using therapeutic doses (5). This study was supported in part by grant 88/1483 from Fondo de Investigaciones Sanitarias from Ministerio de Sanidad y Consumo, Spain. J.V.M.S. is fellow on the Instituto de Salud Carlos III and R.L. is fellow of the Plan Nacional de Formacion de Personal Investigador, Ministerio de Educaci6n y Ciencia, Spain. We acknowledge B. G. Spratt for the revised English version of the manuscript. LITERATURE CITED 1. Biswass, G. D., T. Sox, E. Blackman, and P. F. Sparling. 1977. Factors affecting genetic transformation of Neisseria gonorrhoeae. J. Bacteriol. 129:983-992. 2. Boquete, M. T., C. Marcos, and J. A. Saez Nieto. 1986. Characterization of Neisseria polysacchareae sp. nov. (Riou, 1983) in previously identified noncapsular strains of Neisseria meningitidis. J. Clin. Microbiol. 23:973-975. 3. Botha, P. 1988. Penicillin-resistant Neisseria meningitidis in Southern Africa. Lancet i:54. 4. Campos, J., P. M. Mendelman, M. V. Sako, D. 0. Chaffin, A. L. Smith, and J. A. Saez Nieto. 1987. Detection of relatively penicillin G-resistant Neisseria meningitidis by disk susceptibil-

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