Reduced Levofloxacin Susceptibility and Tetracycline Resistance in a Clinical Isolate of Haemophilus quentini Identified by 16S rRNA Sequenc
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微生物临床杂志 2005年第10期
Centre of Infection and Department of Microbiology, Queen Mary Hospital, The University of Hong Kong
Department of Clinical Pathology, Kwong Wah Hospital, Hospital Authority, Hong Kong Special Administrative Region, China
ABSTRACT
This paper reports the first case of Haemophilus quentini bacteremia with reduced susceptibility to levofloxacin and resistance to nalidixic acid identified by 16S rRNA sequencing. There was an S84L substitution in gyrA and an S84I substitution in parC. The isolate had coresistance to ampicillin (-lactamase positive) and tetracycline mediated by the tet(B) gene.
CASE REPORT
The patient was a newborn, full-term baby girl. She was delivered by the normal vaginal route with a birth weight of 2.9 kg in a general hospital in Hong Kong. The antenatal history was uncomplicated. There was no history of prolonged rupture of membrane or maternal fever. The baby had an Apgar score of 9 at 1 min. Twelve hours after birth, she developed respiratory distress with insucking of the chest and was admitted to a neonatal intensive care unit. Aspiration from a nasogastric tube revealed a large amount meconium-stained fluid. A chest X ray showed a hyperinflated chest and hazziness over both lung fields. Her total leukocyte count was 34.2 x 109/liter (91% neutrophils). The platelet counts and hemoglobin level were normal. A blood culture was performed. She was resuscitated and empirical intravenous ampicillin and netromycin were commenced. She was put on nasal continuous positive airway pressure for respiratory support. Subsequently, the antimicrobial therapy was changed to intravenous amoxicillin-clavulanic acid and was continued for 7 days. The patient recovered after treatment and was discharged on day 13.
Two days later (1 October 2001), the blood culture bottles were positive for a gram-negative coccobacilli. The bacterium (strain S32F2) was grown on chocolate agar to give colonies of 1 mm in diameter after incubation for 24 h at 37°C in air with 5% CO2, but there was no growth on blood agar under the same incubation conditions. Results of the identification by biochemical methods are shown in Table 1. The API NH system (bioMerieux Vitek) but not the Vitek NHI system (bioMerieux Vitek) gave Haemophilus influenzae as the identification at a high confidence level. The organism was subsequently characterized by PCR and near complete sequencing of the 16S rRNA gene, using primers and a protocol we previously described (5). In brief, the sequences of the PCR products were compared with known 16S rRNA gene sequences in the GenBank database by BLAST searches. The length of the 16S rRNA gene included in the analysis was 1,326 bases (this corresponds to positions 28 to 1353 in the sequence of the H. influenzae strain KW20 Rd 16S rRNA gene under accession no. U32741). The BLAST search showed that the bacterium has sequence 100% identical to Haemophilus quentini (GenBank accession no. AF224307).
Susceptibility of the isolate to seven antimicrobial agents was determined by the broth microdilution method, and the results were interpreted as previously described (3, 6). The MICs and the interpretations were as follows: ampicillin, 2 μg/ml (-lactamase positive); cefuroxime, 0.06 μg/ml (susceptible); cefotaxime, 0.001 μg/ml (susceptible); nalidixic acid, 128 μg/ml (resistant); levofloxacin, 0.25 μg/ml (reduced susceptibility); tetracycline, 16 μg/ml (resistant); and minocycline, 1 μg/ml. Mutations within the quinolone-resistance determining regions (QRDRs) of the gyrA and parC genes were examined by PCR and sequencing as described previously (3). The protocol and primers developed by Aminov et al. were used for detection of tetracycline efflux genes (1). Two mutations were found within the QRDRs, and they led to an S84L substitution in GyrA and an S84I substitution in ParC. PCR was positive for the tet(B) gene.
H. quentini is a new species recently proposed for a group of Haemophilus strains isolated from neonatal and genital tract infections (4). Members of this species comprise part of the bacterial population identified as H. influenzae biotype IV in biochemical tests and were previously referred to as "a cryptic genospecies of Haemophilus" (2, 11, 12). They form a homogenous group with unique multilocus enzyme electrophoresis patterns, outer membrane protein profiles, and fimbrial protein gene sequences (2, 9). In 16S rRNA sequence analysis, they form a monophyletic unit with a closer relation to Haemophilus haemolyticus than to H. influenzae (12).
This study corroborates previous studies that H. quentini cannot be distinguished from H. influenzae by the commonly used biochemical tests and commercial systems (8). Although H. quentini commonly masquerades as H. influenzae biotype IV, a false-negative orthinine decarboxylase reaction such as the one we found for the API system in our isolate could lead to erroneous typing as biotype III, as previously reported for some other commercial systems (8).
This study reports for the first time reduced susceptibility to levofloxacin and nalidixic acid resistance in a tetracycline- and ampicillin-resistant clinical strain of H. quentini. The finding is in contrast to the high degree of susceptibilities reported previously for this genospecies (10). Presumably, the strain was transmitted from the mother to the baby. Since both fluoroquinolones and tetracyclines are contraindicated during pregnancy, the finding may indicate that antibiotic resistance of both types could persist for months without a constant selection pressure (13). In our strain, there were two amino acid changes in the QRDRs of GyrA and ParC. While this represents the first report of such in H. quentini, both amino acid substitutions have been previously reported in H. influenzae strains with reduced susceptibility to levofloxacin and resistance to nalidixic acid (7). As the fluoroquinolones group of antibiotic is contraindicated in the neonate, identification of reduced susceptibility to levofloxacin in the present case would mean little in terms of the treatment choices. However, the finding would still carry implications in other patients such as women with genital infections in whom the fluoroquinolones may be used as therapies.
In conclusion, the present study shows that 16S rRNA is useful in identification of newly recognized bacterial species. Our finding indicates the potential for this new species to acquire resistance to multiple antibiotics.
REFERENCES
Aminov, R. I., J. C. Chee-Sanford, N. Garrigues, B. Teferedegne, I. J. Krapac, B. A. White, and R. I. Mackie. 2002. Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria. Appl. Environ. Microbiol. 68:1786-1793.
Gousset, N., A. Rosenau, P. Y. Sizaret, and R. Quentin. 1999. Nucleotide sequences of genes coding for fimbrial proteins in a cryptic genospecies of Haemophilus spp. isolated from neonatal and genital tract infections. Infect. Immun. 67:8-15.
Ho, P. L., K. H. Chow, G. C. Mak, K. W. Tsang, Y. L. Lau, A. Y. Ho, E. L. Lai, and S. S. Chiu. 2004. Decreased levofloxacin susceptibility in Haemophilus influenzae in children, Hong Kong. Emerg. Infect. Dis. 10:1960-1962.
Kilian, M. Genus Haemophilus, p. 883-903. In N. R. Krieg, J. T. Staley, and G. M. Garrity (ed.), Bergey's manual of systematic bacteriology. Springer, New York, N.Y., in press.
Lau, S. K., P. C. Woo, M. Y. Mok, J. L. Teng, V. K. Tam, K. K. Chan, and K. Y. Yuen. 2004. Characterization of Haemophilus segnis, an important cause of bacteremia, by 16S rRNA gene sequencing. J. Clin. Microbiol. 42:877-880.
National Committee for Clinical Laboratory Standards. 2002. Performance standards for antimicrobial susceptibility testing: twelfth informational supplement. M100-S12. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Perez-Vazquez, M., F. Roman, B. Aracil, R. Canton, and J. Campos. 2004. Laboratory detection of Haemophilus influenzae with decreased susceptibility to nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin due to GyrA and ParC mutations. J. Clin. Microbiol. 42:1185-1191.
Quentin, R., I. Dubarry, C. Martin, B. Cattier, and A. Goudeau. 1992. Evaluation of four commercial methods for identification and biotyping of genital and neonatal strains of Haemophilus species. Eur. J. Clin. Microbiol. Infect. Dis. 11:546-549.
Quentin, R., A. Goudeau, R. J. Wallace, Jr., A. L. Smith, R. K. Selander, and J. M. Musser. 1990. Urogenital, maternal and neonatal isolates of Haemophilus influenzae: identification of unusually virulent serologically non-typable clone families and evidence for a new Haemophilus species. J. Gen. Microbiol. 136:1203-1209.
Quentin, R., N. Koubaa, B. Cattier, M. Gavignet, and A. Goudeau. 1988. In vitro activities of five new quinolones against 88 genital and neonatal Haemophilus isolates. Antimicrob. Agents Chemother. 32:147-149.
Quentin, R., C. Martin, J. M. Musser, N. Pasquier-Picard, and A. Goudeau. 1993. Genetic characterization of a cryptic genospecies of Haemophilus causing urogenital and neonatal infections. J. Clin. Microbiol. 31:1111-1116.
Quentin, R., R. Ruimy, A. Rosenau, J. M. Musser, and R. Christen. 1996. Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA. J. Clin. Microbiol. 34:1380-1385.
Salyers, A. A., and C. F. Amabile-Cuevas. 1997. Why are antibiotic resistance genes so resistant to elimination Antimicrob. Agents Chemother. 41:2321-2325.(Gannon C. Mak, P. L. Ho, )
Department of Clinical Pathology, Kwong Wah Hospital, Hospital Authority, Hong Kong Special Administrative Region, China
ABSTRACT
This paper reports the first case of Haemophilus quentini bacteremia with reduced susceptibility to levofloxacin and resistance to nalidixic acid identified by 16S rRNA sequencing. There was an S84L substitution in gyrA and an S84I substitution in parC. The isolate had coresistance to ampicillin (-lactamase positive) and tetracycline mediated by the tet(B) gene.
CASE REPORT
The patient was a newborn, full-term baby girl. She was delivered by the normal vaginal route with a birth weight of 2.9 kg in a general hospital in Hong Kong. The antenatal history was uncomplicated. There was no history of prolonged rupture of membrane or maternal fever. The baby had an Apgar score of 9 at 1 min. Twelve hours after birth, she developed respiratory distress with insucking of the chest and was admitted to a neonatal intensive care unit. Aspiration from a nasogastric tube revealed a large amount meconium-stained fluid. A chest X ray showed a hyperinflated chest and hazziness over both lung fields. Her total leukocyte count was 34.2 x 109/liter (91% neutrophils). The platelet counts and hemoglobin level were normal. A blood culture was performed. She was resuscitated and empirical intravenous ampicillin and netromycin were commenced. She was put on nasal continuous positive airway pressure for respiratory support. Subsequently, the antimicrobial therapy was changed to intravenous amoxicillin-clavulanic acid and was continued for 7 days. The patient recovered after treatment and was discharged on day 13.
Two days later (1 October 2001), the blood culture bottles were positive for a gram-negative coccobacilli. The bacterium (strain S32F2) was grown on chocolate agar to give colonies of 1 mm in diameter after incubation for 24 h at 37°C in air with 5% CO2, but there was no growth on blood agar under the same incubation conditions. Results of the identification by biochemical methods are shown in Table 1. The API NH system (bioMerieux Vitek) but not the Vitek NHI system (bioMerieux Vitek) gave Haemophilus influenzae as the identification at a high confidence level. The organism was subsequently characterized by PCR and near complete sequencing of the 16S rRNA gene, using primers and a protocol we previously described (5). In brief, the sequences of the PCR products were compared with known 16S rRNA gene sequences in the GenBank database by BLAST searches. The length of the 16S rRNA gene included in the analysis was 1,326 bases (this corresponds to positions 28 to 1353 in the sequence of the H. influenzae strain KW20 Rd 16S rRNA gene under accession no. U32741). The BLAST search showed that the bacterium has sequence 100% identical to Haemophilus quentini (GenBank accession no. AF224307).
Susceptibility of the isolate to seven antimicrobial agents was determined by the broth microdilution method, and the results were interpreted as previously described (3, 6). The MICs and the interpretations were as follows: ampicillin, 2 μg/ml (-lactamase positive); cefuroxime, 0.06 μg/ml (susceptible); cefotaxime, 0.001 μg/ml (susceptible); nalidixic acid, 128 μg/ml (resistant); levofloxacin, 0.25 μg/ml (reduced susceptibility); tetracycline, 16 μg/ml (resistant); and minocycline, 1 μg/ml. Mutations within the quinolone-resistance determining regions (QRDRs) of the gyrA and parC genes were examined by PCR and sequencing as described previously (3). The protocol and primers developed by Aminov et al. were used for detection of tetracycline efflux genes (1). Two mutations were found within the QRDRs, and they led to an S84L substitution in GyrA and an S84I substitution in ParC. PCR was positive for the tet(B) gene.
H. quentini is a new species recently proposed for a group of Haemophilus strains isolated from neonatal and genital tract infections (4). Members of this species comprise part of the bacterial population identified as H. influenzae biotype IV in biochemical tests and were previously referred to as "a cryptic genospecies of Haemophilus" (2, 11, 12). They form a homogenous group with unique multilocus enzyme electrophoresis patterns, outer membrane protein profiles, and fimbrial protein gene sequences (2, 9). In 16S rRNA sequence analysis, they form a monophyletic unit with a closer relation to Haemophilus haemolyticus than to H. influenzae (12).
This study corroborates previous studies that H. quentini cannot be distinguished from H. influenzae by the commonly used biochemical tests and commercial systems (8). Although H. quentini commonly masquerades as H. influenzae biotype IV, a false-negative orthinine decarboxylase reaction such as the one we found for the API system in our isolate could lead to erroneous typing as biotype III, as previously reported for some other commercial systems (8).
This study reports for the first time reduced susceptibility to levofloxacin and nalidixic acid resistance in a tetracycline- and ampicillin-resistant clinical strain of H. quentini. The finding is in contrast to the high degree of susceptibilities reported previously for this genospecies (10). Presumably, the strain was transmitted from the mother to the baby. Since both fluoroquinolones and tetracyclines are contraindicated during pregnancy, the finding may indicate that antibiotic resistance of both types could persist for months without a constant selection pressure (13). In our strain, there were two amino acid changes in the QRDRs of GyrA and ParC. While this represents the first report of such in H. quentini, both amino acid substitutions have been previously reported in H. influenzae strains with reduced susceptibility to levofloxacin and resistance to nalidixic acid (7). As the fluoroquinolones group of antibiotic is contraindicated in the neonate, identification of reduced susceptibility to levofloxacin in the present case would mean little in terms of the treatment choices. However, the finding would still carry implications in other patients such as women with genital infections in whom the fluoroquinolones may be used as therapies.
In conclusion, the present study shows that 16S rRNA is useful in identification of newly recognized bacterial species. Our finding indicates the potential for this new species to acquire resistance to multiple antibiotics.
REFERENCES
Aminov, R. I., J. C. Chee-Sanford, N. Garrigues, B. Teferedegne, I. J. Krapac, B. A. White, and R. I. Mackie. 2002. Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria. Appl. Environ. Microbiol. 68:1786-1793.
Gousset, N., A. Rosenau, P. Y. Sizaret, and R. Quentin. 1999. Nucleotide sequences of genes coding for fimbrial proteins in a cryptic genospecies of Haemophilus spp. isolated from neonatal and genital tract infections. Infect. Immun. 67:8-15.
Ho, P. L., K. H. Chow, G. C. Mak, K. W. Tsang, Y. L. Lau, A. Y. Ho, E. L. Lai, and S. S. Chiu. 2004. Decreased levofloxacin susceptibility in Haemophilus influenzae in children, Hong Kong. Emerg. Infect. Dis. 10:1960-1962.
Kilian, M. Genus Haemophilus, p. 883-903. In N. R. Krieg, J. T. Staley, and G. M. Garrity (ed.), Bergey's manual of systematic bacteriology. Springer, New York, N.Y., in press.
Lau, S. K., P. C. Woo, M. Y. Mok, J. L. Teng, V. K. Tam, K. K. Chan, and K. Y. Yuen. 2004. Characterization of Haemophilus segnis, an important cause of bacteremia, by 16S rRNA gene sequencing. J. Clin. Microbiol. 42:877-880.
National Committee for Clinical Laboratory Standards. 2002. Performance standards for antimicrobial susceptibility testing: twelfth informational supplement. M100-S12. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Perez-Vazquez, M., F. Roman, B. Aracil, R. Canton, and J. Campos. 2004. Laboratory detection of Haemophilus influenzae with decreased susceptibility to nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin due to GyrA and ParC mutations. J. Clin. Microbiol. 42:1185-1191.
Quentin, R., I. Dubarry, C. Martin, B. Cattier, and A. Goudeau. 1992. Evaluation of four commercial methods for identification and biotyping of genital and neonatal strains of Haemophilus species. Eur. J. Clin. Microbiol. Infect. Dis. 11:546-549.
Quentin, R., A. Goudeau, R. J. Wallace, Jr., A. L. Smith, R. K. Selander, and J. M. Musser. 1990. Urogenital, maternal and neonatal isolates of Haemophilus influenzae: identification of unusually virulent serologically non-typable clone families and evidence for a new Haemophilus species. J. Gen. Microbiol. 136:1203-1209.
Quentin, R., N. Koubaa, B. Cattier, M. Gavignet, and A. Goudeau. 1988. In vitro activities of five new quinolones against 88 genital and neonatal Haemophilus isolates. Antimicrob. Agents Chemother. 32:147-149.
Quentin, R., C. Martin, J. M. Musser, N. Pasquier-Picard, and A. Goudeau. 1993. Genetic characterization of a cryptic genospecies of Haemophilus causing urogenital and neonatal infections. J. Clin. Microbiol. 31:1111-1116.
Quentin, R., R. Ruimy, A. Rosenau, J. M. Musser, and R. Christen. 1996. Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA. J. Clin. Microbiol. 34:1380-1385.
Salyers, A. A., and C. F. Amabile-Cuevas. 1997. Why are antibiotic resistance genes so resistant to elimination Antimicrob. Agents Chemother. 41:2321-2325.(Gannon C. Mak, P. L. Ho, )