J. Dairy Sci. 2008. 91:4075-4081. doi:10.3168/jds.2008-1040
© 2008 American Dairy Science Association ®
Identification and Antimicrobial Resistance of Streptococcus uberis and Streptococcus parauberis Isolated from Bovine Milk Samples
A. Pitkälä*,1,
J. Koort
and
J. Björkroth
* Finnish Food Safety Authority Evira, Mustialankatu 3, FIN-00790 Helsinki, Finland
Faculty of Veterinary Medicine, PO Box 66 (Agnes Sjöbergin katu 2), 00014 Helsinki University, Finland
1 Corresponding author: anna.pitkala{at}evira.fi
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ABSTRACT
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The conventional identification of Streptococcus uberis/parauberis group (n = 137) in clinical and subclinical bovine mastitis samples originating from 111 different farms was compared with identification based on 16 and 23S rRNA gene HindIII RFLP patterns used as operational taxonomic units in numerical analyses. On the basis of ribopattern analysis only 2 isolates belonged to S. parauberis, which is thus not a frequent cause of bovine intramammary infections in Finland. According to in vitro antimicrobial susceptibility testing, Streptococcus uberis is susceptible to β-lactam antibiotics. The prevalence of erythromycin (15.6%) and oxytetracycline (40.6%) resistance of clinical S. uberis isolates was higher than reported previously among subclinical isolates. The 2 subclinical S. parauberis isolates were susceptible to all the antimicrobials tested.
Key Words: mastitis • identification • resistance • Streptococcus species
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INTRODUCTION
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Environmental streptococci, including Streptococcus uberis, have emerged as important mastitis pathogens in different areas of the world. Although implementation of mastitis control strategies has reduced the number of clinical mastitis cases caused by contagious pathogens, the importance of S. uberis as a mastitis pathogen has increased, and it is a significant problem in, for example, New Zealand (Douglas et al., 2000), the United Kingdom (Bradley, 2002), and the United States (Wilson et al., 1997). In Finland, S. uberis is the third commonest finding in both clinical and subclinical mastitis samples (Koivula et al., 2007). The definition of environmental streptococci is variable, and non-β-hemolytic, catalase-negative, gram-positive cocci include several species of esculin-positive streptococci, enterococci, lactococci, and aerococci (Fortin et al., 2003). Correct identification to the species level is important for the choice of proper treatment because these species have different pathogenicities and different antibiotic susceptibility profiles. Furthermore, accurate identification is a necessity for the development and modification of control methods (Devriese et al., 1999; Rossitto et al., 2002).
The conventional tests routinely used for identification of gram-positive, catalase-negative cocci can lead to misidentification (Fortin et al., 2003; Bosshard et al., 2004). For example, using conventional microbiological methods, S. uberis and S. parauberis are nearly indistinguishable (Facklam, 2002). The only phenotypic criterion allowing differentiation is the production of β-D-glucuronidase by S. uberis (Khan et al., 2003). During the last few decades, DNA-based methods such as species-specific oligonucleotide probes (Bentley et al., 1993; Harland et al., 1993), PCR-based methods with species-specific primers (Hassan et al., 2001; Alber et al., 2004) or RFLP (Jayarao et al., 1991, McDonald et al., 2005), and 16S and 23S rRNA gene RFLP patterns (ribotypes; Williams and Collins, 1991; Koort et al., 2006) have been used to differentiate between S. uberis and S. parauberis. In addition to the differentiation between S. uberis and S. parauberis, numerical analysis of RFLP patterns, used as operational taxonomic units, has been found reliable for the species level identification in several genera of gram-positive, catalase-negative cocci, when used with validated ribopattern database (Rinkinen et al., 2004; Björkroth et al., 2005; 
vec et al., 2005).
The possible misidentification of environmental streptococci affects the reliability of antibiotic resistance reports. For example, although S. uberis is usually susceptible to penicillin, there are reports of reduced susceptibility or resistance to penicillin in environmental streptococci or S. uberis (Erskine et al., 2002; Guérin-Faublée et al., 2002; Rajala-Shultz et al., 2004). It is not always clear whether the isolates tested have been validly differentiated from other aesculin-positive cocci, such as Enterococcus spp., Aerococcus viridans, or Lactococcus spp., which have different antimicrobial susceptibility profiles.
In this study, we first confirmed the species level identification of putative S. uberis isolated from subclinical and clinical mastitis samples in Finland, and determined the frequency of S. parauberis among them. Some studies on the frequency of S. parauberis were already conducted in Germany, the Netherlands, and Australia (Khan et al., 2003; MARAN, 2003; McDonald et al., 2005) where the average herd size is 3 to 10 times of that in Finland. Also, the antimicrobial usage in dairy herds with possible relationship to bacterial resistance varies between different countries. We therefore also determined the antimicrobial resistance of S. uberis isolated from clinical samples in Finland.
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MATERIALS AND METHODS
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Bacterial Isolates
The material consisted of 137 subclinical and clinical isolates identified as Streptococcus uberis by the phenotypic methods described below. The 73 subclinical isolates originated from quarter milk samples (from 1 to 5 isolates per herd) collected during a mastitis survey conducted in Finland in 2001 (Pitkälä et al., 2004). In that study, a quarter was considered to be subclinical if 
500 cfu/mL of bacteria was cultured from the quarter milk sample, when healthy cows from randomly selected herds were sampled. The 64 clinical isolates originated from clinically diseased quarters with abnormal changes in milk. These milk samples were collected in 2005 (one isolate/herd) and were received from mastitis laboratories as pure cultures. All the isolates included in this study were confirmed according to methodology based on Honkanen-Buzalski and Seuna (1995) and NMC (1999) and accredited according to EN ISO/IEC standard 17025 (ISO, 2005). The identification was based on characteristic colony morphology, hemolysis, gram-staining, catalase test, hydrolysis of aesculin, penicillin tolerance test, hydrolysis of hippurate, and production of acid from inulin, raffinose, and sorbitol as a standard confirmation. In cases of doubtful identification, commercial biochemical tests (API Strep, BioMérieux, Marcy l’Etoile, France) were also used. Streptococcus uberis ATCC 9927, Streptococcus bovis ATCC 9809, Enterococcus faecalis ATCC 29212, Lactococcus lactis ATCC 19435, and Aerococcus viridans ATCC 700406 were used as control strains in conventional identification.
Ribotyping
For ribotyping, chromosomal DNA was isolated by using the guanidium thiocyanate method of Pitcher et al. (1989) as modified by Björkroth and Korkeala (1996) by combining lysozyme (25 mg/mL, Sigma, St, Louis, MO) and mutanolysin (200 U/mL, Sigma) lysis of bacterial cells. Digestion of DNA with HindIII enzyme, restriction enzyme analysis and Southern blotting were performed as described previously (Björkroth and Korkeala, 1996). The cDNA probe for ribotyping was reverse-transcripted using Escherichia coli MRE600 16 and 23S rRNA (Roche Molecular Biochemicals, Mannheim, Germany) as a template and labeled as described previously by Blumberg et al. (1991). Membranes were hybridized at 58°C overnight, and detection of the digoxigenin label was performed as recommended by Roche Molecular Biochemicals. Scanned ribopatterns were analyzed using the BioNumerics 4.5 software package. The similarity between all pairs was expressed by the Dice coefficient correlation, and UPGMA clustering was used for construction of the dendrograms. Based on the use of internal control in the database, pattern optimization and band position tolerance of 0.7 and 1.5, respectively, were allowed.
Antimicrobial Susceptibility Testing
All 64 clinical isolates, confirmed as S. uberis by the results of the numerical analysis of ribopatterns, were studied for their susceptibility to antimicrobials using a commercially available microdilution system (VetMIC, SVA, Uppsala, Sweden) according to the manufacturer’s instructions and by following the standards of the Clinical and Laboratory Standards Institute (CLSI, 2002). Before testing, the isolates were subcultured onto blood agar and incubated for 24 h at 37°C. To test the susceptibility of streptococcal isolates, Mueller Hinton broth (Difco, le Port de Claix, France) was supplemented with 7% defibrinated horse serum as described in the manufacturer’s instructions. Minimum inhibitory concentration was recorded as the lowest concentration of the antimicrobial agent that inhibited bacterial growth. The distributions of the MIC were calculated, and the results were classified using the breakpoints recommended by the CLSI (2004) for bacteria isolated from animals. For clindamycin, we used the breakpoint used with mastitis pathogens by Gianneechini et al. (2002). For isolates that were classified as both erythromycin resistant and clindamycin susceptible, erm-mediated inducible clindamycin resistance was determined by the double-disk D-test on a standard blood agar plate with a 15-µg erythromycin disc and a 2-µg clindamycin disc (Rosco, Taastrup, Denmark; CLSI, 2007). The differences in distribution to ribotypes and resistance patterns were compared using the 2-sample proportion test in the statistical software package Statistix (Analytical Software, Tallahassee, FL).
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RESULTS
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All 137 isolates were classified to Streptococcus species according to the penicillin tolerance test (Honkanen-Buzalski and Seuna, 1995). Of these isolates, 132 were directly confirmed as S. uberis with conventional methods because they hydrolyzed aesculin and hippurate, and produced acid from inulin and sorbitol but not from raffinose. Five isolates showed atypical reactions in the conventional methods: 2 isolates were inulin negative, 1 aesculin negative, 1 raffinose positive, and 1 both inulin and hippurate negative. Two of these isolates, inulin negative isolate 2685 and raffinose positive isolate 153–66, are presented in Figure 1
. However, with API Strep the species identification of these isolates to S. uberis was excellent, very good, or good (with probability of species identification of 99.9% with a T value of 0.75; 99.0% with a T value of 0.5 and 90% with a T value of 0.25, respectively, with probability of identification and T being manufacturer-defined variables). To confirm the conventional identification of all these 137 isolates and to differentiate between S. uberis and S. parauberis, the 16S and 23S HindIII RFLP patterns (HindIII ribopatterns) of the isolates were analyzed. HindIII restriction enzyme was chosen because it has been found to provide species-specific patterns for various bacteria (Björkroth and Korkeala, 1996; Koort et al., 2005, 2006). Numerical analysis of the patterns, functioning as operational taxonomic units, results in cluster formation. Based on the locations of the type and reference strains in these clusters, isolates are considered to represent corresponding species. The ribopattern database (the Department of Food and Environmental Hygiene, University of Helsinki, Finland) against which the patterns were compared contains HindIII patterns of approximately 300 type and reference strains and over 6,000 other isolates (Susiluoto et al., 2003; Koort et al., 2005; Vihavainen et al., 2007). Figure 1 shows the selected streptococcal type and reference strains, which were included in comparisons. By ribotype analysis, 135 isolates were confirmed to be S. uberis (cluster I, pattern similarities within 72%). Within the S. uberis cluster (cluster I) there were 30 different ribotypes, 1 to 40 isolates in each. Table 1 shows the division of the S. uberis isolates into different ribotypes. In one ribotype, RT 7, the number of clinical isolates is statistically higher than the number of subclinical isolates (P < 0.01), but the number of isolates is low (Table 1). Two of the subclinical isolates clearly differentiated from S. uberis strains and were identified as S. parauberis by the numerical analysis (cluster II, 100% pattern similarity, and cluster III, pattern similarities 85%). The phenotypic reactions of these 2 isolates were typical for S. uberis in conventional microbiological tests. The API Strep could not reliably identify them, but the identification was either "low discrimination" (Lactococcus lactis ssp. lactis/Enterococcus faecalis/Enterococcus faecium, %id of 69.2% with a T value of 0.44, 17.2% with a T value of 0.21, and 11.9% with a T value of 0.33, respectively) or "doubtful profile" (S. uberis, %id 96.9% with a T value of 0.25). Figure 1 shows the species-specific clusters obtained in numerical analysis of 16 and 23 S rRNA gene HindIII RFLP patterns (ribotypes). Within each cluster, only a representative set of different ribopatterns, originating from both clinical and subclinical isolates, is represented.
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Figure 1. Numerical analysis of 16 and 23S RFLP patterns (ribotypes) generated by HindIII enzyme. All the isolates within the cluster I are identified as Streptococcus uberis, and within the clusters II and III as Streptococcus parauberis.
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The in vitro activities of the antimicrobials tested are presented in Table 2 for clinical S. uberis isolates. For comparison, also the susceptibility results of subclinical isolates published in Pitkälä et al. (2004) are included in Table 2. All the S. uberis and S. parauberis isolates were sensitive to penicillin (MIC of S. parauberis 0.03 and 0.12 µg/mL). The MIC50 and MIC90 of the S. uberis isolates for penicillin were both 0.064 µg/ mL. All the clinical S. uberis isolates and both of the S. parauberis isolates were also sensitive to cephalothin and clindamycin (MIC of S. parauberis 0.5 and 1 µg/ mL, respectively). Of the S. uberis isolates, 40.6% were resistant to oxytetracycline and 15.6% were resistant to erythromycin. Nine of the 10 erythromycin-resistant S. uberis isolates were also resistant to tetracycline. Eight of these isolates were included in 1 ribotype (RT 7), whereas the isolates with resistance for only tetracycline were distributed into 8 ribotypes, most of them (7/12) into RT 10 (Table 1
). Both of the S. parauberis isolates were sensitive to oxytetracycline and erythromycin (MIC 1 and 0.25 µg/mL, respectively). No multiresistance was detected in either species. With the D-test, none of the erythromycin-resistant isolates showed inducible resistance to clindamycin.
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DISCUSSION
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According to our results, S. parauberis is not a frequent cause of bovine intramammary infections in Finland. This species has also been shown in other studies to be less common in bovine mastitis than previously believed. In Germany, only 1 of 131 Streptococcus isolates was classified as S. parauberis and 130 as S. uberis (Khan et al., 2003). In the Netherlands, 2 of 83 S. uberis isolates were identified as S. parauberis (MARAN, 2003). In Australia, 6 of 57 strains conventionally identified as S. uberis were classified as S. parauberis, 2 strains as the closely related Streptococcus iniae, and 2 strains as Aerococcus viridans (McDonald et al., 2005).
In routine diagnostic laboratories, which have no molecular or even commercial phenotypic identification systems available, the identification scheme should still be sufficient to distinguish all gram-positive catalase-negative cocci isolated from mastitis cases (Devriese et al., 1999; Fortin et al., 2003; Odierno et al., 2006). The conventional identification scheme used here, the standard procedure complemented with API 20 Strep when needed, appears to be sufficient to differentiate S. uberis/parauberis as a group from other gram-positive, catalase-negative cocci. However, even Api 20 Strep could not differentiate between S. uberis and S. parauberis, which reflect the lack of updating of the associated database (Hoshino et al., 2005).
The elevated or bimodal distribution of penicillin MIC, which has been reported elsewhere (Guérin-Faublée et al., 2002, Rossitto et al., 2002; MARAN, 2005), was not observed here. The MIC50 and MIC90 values of 0.064 µg/mL in this study were lower than reported in other studies. Rossitto et al. (2002) reported MIC50 and MIC 90 values for penicillin of 0.25 µg/mL, Pol and Ruegg (2007) reported 0.12 µg/mL, and Guérin-Faublée et al. (2003) reported 0.03 µg/mL and 0.25 µg/ mL, respectively, but they also tested lower dilutions than in our study.
The prevalences of erythromycin (15.6%) and tetracycline (40.6%) resistance observed here among the clinical isolates are similar to or slightly lower than those reported previously (Erskine et al., 2002; Guérin-Faublée et al., 2002; Rossitto et al., 2002). However, these results of clinical isolates, which differ significantly (P < 0.01) from earlier Finnish results, are contrary to the expectation. In a survey of subclinical mastitis conducted in 2001 (Pitkälä et al., 2004), only 1% of S. uberis isolates were resistant to oxytetracycline, and all isolates were susceptible to erythromycin using the same techniques in both studies. Furthermore, the use of tetracyclines in mastitis therapy has decreased since the beginning of the 1990s. We have not found any comparison of resistance between clinical and subclinical isolates of S. uberis in the literature. Gianneechini et al. (2002) found no erythromycin or tetracycline resistance in S. uberis isolated from clinical or subclinical mastitis samples, and Roesch et al. (2006) found 10.5% erythromycin resistance but no tetracycline resistance in S. uberis isolated from clinical mastitis samples from cows in integrated or organic production, but the number of tested isolates in both studies was small. Tomita et al. (2008) found differences between subclinical and clinical isolates with MLST and PFGE methods and the presence of hasA/hasC (genes regulating capsulation). Unfortunately, they did not test the resistance patterns of the isolates.
Nine (14.1%) of the S. uberis isolates were resistant to both tetracycline and erythromycin. Interestingly, 8 of these isolates were included in RT 7 (Table 1) in which also the number of clinical isolates was statistically higher than the number of subclinical isolates (P < 0.01). The combined resistance has also been observed in S. pyogenes (Giovanetti et al., 2003; Brenciani et al., 2004, 2007) and in S. pneumoniae (Montanari et al., 2003). Both of the major macrolide resistance mechanisms, drug efflux or methylation of the ribosomal drug binding site has been found among the isolates with combined resistance (Montanari et al., 2003; Brenciani et al., 2004, 2007). Of these mechanisms, drug efflux resistance results in low level resistance to erythromycin but not to clindamycin, which is the resistance pattern seen here.
In conclusion, S. parauberis is not a frequent cause of bovine intramammary infections in Finland, and it is not essential to differentiate between S. uberis and S. parauberis in mastitis diagnostics. The DNA-based methods should be utilized for studying the epidemiology and pathogenesis of S. uberis mastitis, as well as the genetic background of resistance because these matters require further studies. Penicillin is effective in treatment of clinical mastitis caused by S. uberis in Finland. However, regular resistance monitoring is needed to monitor changes in resistance patterns.
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ACKNOWLEDGEMENTS
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The authors gratefully acknowledge the excellent technical assistance of Henna Niinivirta and Erja Merivirta at the University of Helsinki and Noora Lehtonen, Kaisa Rouhiainen, and Kaija Tornberg at the Finnish Food Safety Authority Evira. J. Björkroth’s group is grateful to the Academy of Finland for its financial support through the national programme for Centre of Excellence in Microbial Food Safety Research.
Received for publication January 22, 2008.
Accepted for publication June 2, 2008.
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ABSTRACT
INTRODUCTION
