Multiple Resistance to Betalactam Antibiotics

Clin. Lab. 2013;59:381-387 ©Copyright

ORIGINAL ARTICLE

Multiple Resistance to Betalactam Antibiotics, Azithromycin or Moxifloxacin in Implant Associated Bacteria JULIA KARBACH 1, ANGELIKA S. CALLAWAY 2, BRITA WILLERSHAUSEN 2, WILFRIED WAGNER 1, BILAL AL-NAWAS 1 1

Department for Oral and Maxillofacial Surgery, University Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany 2 Department of Operative Dentistry, University Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany

SUMMARY Background: Antibiotics are more and more frequently prescribed in dentistry for prevention and treatment of oral diseases. Bacterial resistance to these agents is clearly increasing, including even previously susceptible micro-organisms and true pathogens. The aim of the present investigation was to examine resistant bacterial strains with respect to possible multiple antibiotic resistance. Methods: In a previous investigation, implant-associated bacteria were tested first as mixed cultures and again as pure isolates (n = 138) for resistance to one of five antibiotics (ampicillin/AM, ampicillin + sulbactam/AB, azithromycin/AZ, penicillin/PG, moxifloxacin/MX) using the Etest®. The resistance of most of the pure isolates was lower than in mixed culture, but 31.2% had retained their original resistance. Subsequently, all 138 isolates were tested for resistance or susceptibility to the other four antibiotics, again using the Etest®. Results: 27.6% (38/138) of the isolates retained their original antibiotic resistance and were resistant to at least one other antibiotic (MIC ≥ 128 μg/mL for AB, AM or AZ, ≥ 32 μg/mL PG, ≥ 24 μg/mL MX). 2.2% (3/138) strains had lost their original antibiotic resistance, but were resistant to at least one other antibiotic (MIC ≥ 128 μg/mL for AB, AM, or AZ, ≥ 32 μg/mL PG, ≥ 24 μg/mL MX). Conclusions: Some of the isolates belonging to the implant-associated microflora were multi-resistant, even though the patients had not received any antibiotics six weeks prior to the sampling. The exact mechanisms that lead to multiple resistance need to be examined in further studies. (Clin. Lab. 2013;59:381-387. DOI: 10.7754/Clin.Lab.2012.120422) ics and increased resistance of bacteria could be seen when comparing the antibiotic use (including amount and type) in different countries. In the presence of antibiotics a selective pressure on the environment occurs and bacterial resistance to these agents are increasing, involving even previously susceptible micro-organisms, including true pathogens [3]. These resistant micro-organisms create a risk of ineffective therapy, which can lead to an increase in morbidity and mortality of patients. However, the role of resistant bacteria and particularly multi-resistant bacteria of oral infections has received relatively little attention. No data concerning antibiotic resistance are available about infections of soft and hard tissues surrounding dental implants. Non treatment of peri-implant mucositis (re_____________________________________________

KEY WORDS antibiotic resistance, implant associated bacterial flora INTRODUCTION Antibiotics are more and more frequently prescribed in dentistry for the prevention and treatment of oral diseases. Outside dentistry, most antibiotics are used for therapy including otitis media, upper respiratory tract infections, bronchitis, pharyngitis and sinusitis [1]. Clinicians should choose antibiotics on a case-specific basis, and the choice should be based on several factors, such as laboratory data, patient’s health, age, allergies, drug absorption, and plasma levels [2]. A direct relationship between frequency of prescriptions of antibiot-

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J. KARBACH et al. Table 1. Multiple resistant bacteria from the peri-implant sulcus, originally resistant to azithromycin (≥ 128 µg/mL), moxifloxacin (≥ 32 µg/mL) or penicillin G (≥ 32 µg/mL), using the Etest. One strain (Parvimonas micra) had lost its original antibiotic resistance, but was resistant to two other antibiotics; another strain (Haemophilus species) had lost its original antibiotic resistance, but was resistant to three other antibiotics. azithromycin

moxifloxacin

penicillin G

ampicillin

ampicillin/ sulbactam

Genus or Species

256

32

32

256

256

Streptococcus species

256

32

32

256

256

Staphylococcus aureus

256

0.75

32

256

256


256

32

0.047

0.125

0.125

Parvimonas micra

256

32

32

256

256


256

32

32

256

256


256

0.125

32

256

256

Streptococcus species*

256

32

32

256

256


128

32

32

256

256


128

24

32

256

128

Acinetobacter species

0.19

32

0.094

32

256

Parvimonas micra

azithromycin

moxifloxacin

penicillin G

ampicillin


256

32

32

256

256


32

3

32

256

256

Haemophilus species

azithromycin

moxifloxacin

penicillin G

ampicillin


32

0.5

32

256

8

Enterobacteriaceae

2

32

32

4

2

Lactobacillus species*

256

32

32

2

256


256

32

32

256

256

Lactobacillus species*

* Isolated from patient no 22 with clinical signs of peri-implant mucositis.

versible inflammation of the soft tissues surrounding implants), can lead to peri-implantitis (inflammation of the soft tissue combined with loss of bone [4]), which can result in the loss of the implant. Implant loss due to peri-implantitis may be kept to a minimum if the lesions are detected and treated at an initial stage [5]. Prior to the use of antibiotics in periimplant therapy, only a few periodontal pathogens are normally detected by microbial testing [6,7]. The choice of the microbiological test (culture, immunoassays, enzyme tests, or nucleic acid based techniques) depends on the intended result. Culture techniques are convenient for estimating the antimicrobial resistance. An optimized antimicrobial treatment is indicated for each case where antibacterial therapy is required [8]. The aim of the present in vitro investigation was to examine bacterial strains, selected from the total implant-associated micro-flora, for possible multiple antibiotic resistance.

MATERIALS AND METHODS In a previous investigation, implant-associated bacteria from 24 patients with dental implants were sampled and tested first as mixed cultures and again as pure isolates (n = 138) for resistance to one of five antibiotics (ampicillin/AM, ampicillin + sulbactam/AB, azithromycin/ AZ, penicillin/PG, moxifloxacin/MX) using the Etest®, as previously described [9]. Inclusion criteria for the subjects were no antibiotic therapy six weeks prior to the examination and no use of antibacterial mouthwash 24 hours before the examination. The resistance of most of the pure isolates, identified at least to the genus level as previously described in detail [9] was lower than in mixed culture, but 31.2% had retained their original resistance. Subsequently, all 138 isolates were tested for resistance or susceptibility to the other four antibiotics, again using the Etest®.

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ANTIBIOTIC RESISTANCE IN IMPLANT ASSOCIATED BACTERIA Table 2. Multiple resistant bacteria from the peri-implant sulcus, originally resistant to ampicillin or ampicillin/sulbactam (≥ 256 µg/mL), using the Etest. One strain (Streptococcus species) had lost its original antibiotic resistance, but was resistant to the four other antibiotics. azithromycin

moxifloxacin

penicillin G

ampicillin


Bacterium

32

32

32

256

12

Lactobacillus species

32

12

32

256

4


256

32

32

256

256


256

32

32

256

256

Acinetobacter species**

32

32

32

256

12

Corynebacterium species

256

0.19

32

256

16

Staphylococcus species

32

32

32

256

256


48

0.38

32

256

12


16

3

32

256

32

Enterobacteriaceae

256

32

32

256

256

Gram-negative coccus*

256

32

32

256

256


8

0.75

32

256

4


32

0.38

32

128

32

Acinetobacter species

256

32

32

4

256


azithromycin

moxifloxacin

penicillin G

ampicillin


256

32

32

256

256


256

32

32

256

256

Lactobacillus paracasei#

64

0.5

32

256

256

short rods

0.25

0.38

32

256

256


96

16

32

256

256

Staphylococcus aureus*

256

32

32

256

256

Haemophilus species

256

0.38

32

256

256

Parvimonas micra*

256

32

32

256

256

A. a.*

256

32

32

256

256

A. a.*

256

0.032

0.047

0.125

256


A. a. = Aggregatibacter actinomycetemcomitans, * Isolated from patient no 22 with clinical signs of peri-implant mucositis, ** Etests of this isolate are shown in Figures 1 and 2 A-E, # Identified by species-specific PCR.

sistant to at least one other antibiotic (MIC ≥ 128 μg/mL for AB, AM or AZ, ≥ 32 μg/mL PG, ≥ 24 μg/mL MX) (Tables 1 and 2; Parvimonas micra, Haemophilus spec., Streptococcus spec.). The majority of the 41 resistant bacteria were Gram-positive cocci or rods (mainly streptococci or lactobacilli) with only a few Gramnegative organisms, including Aggregatibacter actino-

RESULTS 27.6% (38/138) of the isolates retained their original antibiotic resistance and were resistant to at least one other antibiotic (MIC ≥ 128 μg/mL for AB, AM or AZ, ≥ 32 μg/mL PG, ≥ 24 μg/mL MX). 2.2% (3/138) strains had lost their original antibiotic resistance, but were re-

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Figure 1. Mixed cultures tested for resistance to ampicillin showing resistant strains to the highest concentration of the Etest.

Figure 2. Isolate of the mixed culture from ampicillin (Acinetobacter spec.), retaining its original antibiotic resistance to ampicillin (D), and showing resistance to the antibiotic azithromycin (256 µg/mL) (A), moxifloxacin (32 µg/mL) (B), penicillin G (32 µg/mL) (C), and ampicillin/sulbactam (256 µg/mL) (E).

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ANTIBIOTIC RESISTANCE IN IMPLANT ASSOCIATED BACTERIA

were resistant to penicillin G, ampicillin, and moxifloxacin. In a review focussing on antibiotic resistance in food lactic acid bacteria, resistance to penicillin G and ampicillin is reported as a common phenomenon in various lactobacillus species [19]. A possible explanation for the occurrence of resistant lactobacilli in peri-implant sites could be that they are introduced into the oral cavity by way of food intake. In a more recent study about antimicrobial susceptibility of lactobacilli isolated from human blood cultures, resistance to moxifloxacin was reported for lactobacillus species L. gasseri, but not for L. paracasei [20]. In contrast, in the present investigation a strain of L. paracasei was found to be resistant to all five antibiotics, including moxifloxacin. The role of multi-resistant micro-organisms belonging to the healthy flora of the mouth is still unclear. This also raises the question as to whether one strain might provide other bacteria with a resistance mechanism. However, this question was not considered in this investigation. The discovery of Staphylococcus spec. in the peri-implant sulcus has been described by different authors [2123], and a possible link between the occurrence of Staphylococcus aureus and peri-implant infections is currently being discussed. The finding from the present investigation that such strains show a multi-resistant behaviour against the tested antibiotics in both healthy and infected peri-implant sulci might be of great interest in this context. A relationship between antibiotic resistance of the oral micro-flora and the improper use of antibiotics, in terms of either indication or the dosage (duration of treatment being too long or dosage being too weak) has been reported [24]. On the other hand, an increase in prescripttions of antibiotics in dentistry along with an increase in the number of antibiotic resistant bacteria seems to play a role when considering multiple resistant strains [25]. An antibiotic frequently used in periodontal [26] and peri-implant infections is metronidazole, which was not tested in the investigation. Because of the appearance of metronidazole resistant strains of A. a., isolated from patients with periodontal disease [27], a combination of metronidazole with amoxicillin was suggested instead for the therapy of peri-implant infections [28]. Müller et al. 2002 [27] reported that their isolates of A. a. were sensitive to ampicillin + sulbactam, moxifloxacin, and moderately sensitive to azithromycin. In contrast, the two isolates of A. a. from the present investigation were resistant to all five antibiotics, and thus a combination of metronidazole with an aminopenicillin would not be effective. Al-Haroni et al. 2006 [29] found subgingival bacteria with a resistance against ampicillin in 28.9% of Yemeni patients and in 7.9% of Norwegian patients, and against metronidazole in 60.3% and 11.3%, respectively. The number of species resistant to ampicillin and metronidazole was higher in the Yemeni than in the Norwegian samples, which can be associated with the different usage of the two drugs in dental therapy in both countries. In this investigation neither an improper use of antibiot-

mycetemcomitans (A. a.) (Tables 1 and 2). The resistant strains were collected from patients with healthy periimplant mucosa 61.0% (25/41) and from patients with peri-implant mucositis 39.0% (16/41). Resistance to all five antibiotics was seen in 43.9% (18/ 41) of the isolates, and of these strains, six were isolated from a single patient (No. 22) with clinical signs of a peri-implant mucositis 1. In total, 24% (10/41) of the strains were isolated from patient No. 22. Two of the isolates resistant against all five tested antibiotics were identified as A. a.*, two as Acinetobacter spec., one as Haemophilus spec., one as Gram-negative coccus*, one* plus two as Lactobacillus spec., three as Staphylococcus spec., and two* plus four as Streptococcus spec.. Resistance to four of the five antibiotics was seen in 12.1% (5/41) isolates. One isolate was identified as Lactobacillus spec., one as Parvimonas micra*, and one* plus two as Streptococcus spec.. Resistance to three of the five antibiotics was seen in 17.1% (7/41) of the isolates. One isolate belonged to Enterobacteriaceae, one was identified as Corynebacterium spec., one as a short rod, two as Lactobacillus spec., and one* plus one as Staphylococcus spec.. Resistance to two of the five antibiotics was seen in 19.5% (8/41) of the isolates. One isolate was identified as Acinetobacter spec., one as Streptococcus spec., one as Staphylococcus spec., one as Parvimonas micra, one* plus two as Lactobacillus spec., and one belonged to Enterobacteriaceae. (1 strains isolated from the patient (No. 22) are marked with*)

DISCUSSION Multi-resistance was found in bacteria of the peri-implant flora, independent of their pathogenic potential. The existence of antibiotic resistance in bacteria, and even multiple resistance, has been frequently described in literature, including bacteria from the oral cavity associated with various diseases [10-15]. However, antibiotic resistant bacteria are not well known among implant-associated micro-flora. Not every patient in this investigation showed clinical signs of a peri-implant infection, and multi-resistant strains were detected in infected and healthy peri-implant sulci. At the implant sites with clinical signs of a peri-implant mucositis or peri-implantitis, periodontal pathogens like Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Parvimonas micra and others are implicated in the infection [16], despite the fact that these bacteria can be found in lower concentrations in healthy peri-implant sulci [17]. A. a. as well as Parvimonas micra were determined as multi-resistant bacteria in this investigation. Surprisingly, the majority of the multi-resistant isolates were identified as lactobacilli and streptococci, which belong to the oral micro-flora normally associated with oral health [18]. In the present investigation, most lactobacilli with a few exceptions

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J. KARBACH et al. 12. Sedgley CM, Lee EH, Martin MJ, Flannagan SE. Antibiotic resistance gene transfer between Streptococcus gordonii and Enterococcus faecalis in root canals of teeth ex vivo. J Endod 2008;34: 570-4.

ics (exclusion criteria antibiotic therapy six weeks prior to the examination) nor a high prescription rate of the tested antibiotics for azithromycin and moxifloxacin for the therapy of peri-implant infections provides an adequate explanation for the existence of multi-resistant strains. However, good results have been reported when using moxifloxacin as adjunct to scaling and root planing in the therapy of periodontitis [30]. The advantage of a good penetration into tissues and antibacterial activity within cells might be interesting in this context. Bacteria from the normal oral flora could serve as hosts of antibiotic resistance genes. The possible gene transfer between such bacteria and true pathogens in the periimplant sulcus should be considered, and further studies are needed to elucidate the role of resistance of bacteria assosociated with oral health.

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Declaration of Interest: The authors do not have any conflicts of interest.

17. Casado PL, Otazu IB, Balduino A, de Mello W, Barboza EP, Duarte ME. Identification of periodontal pathogens in healthy periimplant sites. Implant Dent 2011;20:226-35.

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Correspondence: Julia Karbach DMD, MD Department for Oral and Maxillofacial Surgery University Medical Centre of the Johannes Gutenberg University Mainz Augustusplatz 2 55131 Mainz, Germany Tel.: +49/6131/173191 Fax: +49/6131/17662 Email: [email protected]

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