Antimicrobial susceptibility patterns for aerobic bacteria isolated from reptilian samples submitted to a veterinary diagnostic laboratory: 129 cases (2005–2016)

Pak Kan Tang 1Royal Veterinary College, North Mymms, AL9 7TA, England
2Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Stephen J. Divers 2Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Susan Sanchez 1Royal Veterinary College, North Mymms, AL9 7TA, England

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Abstract

OBJECTIVE

To identify antimicrobial susceptibility patterns for aerobic bacteria isolated from reptilian samples and, from those patterns, identify antimicrobials that could be considered for empirical treatment of reptiles with suspected bacterial infections.

SAMPLES

129 bacterial isolates from 61 of 127 samples from 96 reptiles.

PROCEDURES

Medical records of reptiles (chelonian, crocodilian, lizard, and snake) presented to the zoological medical service of a veterinary teaching hospital between January 2005 and December 2016 were reviewed for submissions of patient samples for aerobic bacterial culture and susceptibility testing. Sample type, presence or absence of bacterial growth, and antimicrobial susceptibilities of isolated bacteria were recorded. The isolation frequency and the antimicrobial susceptibilities of bacterial genera and species were tabulated.

RESULTS

Pseudomonas spp and Enterococcus spp were the most frequently isolated gram-negative and gram-positive bacteria, respectively. Isolates of gram-negative bacteria frequently had susceptibility to amikacin (86%), gentamicin (95%), tobramycin (92%), and trimethoprim-sulfamethoxazole (83%), and gram-positive bacteria frequently had susceptibility to ampicillin (83%), chloramphenicol (92%), doxycycline (100%), and gentamicin (100%). Isolates of gram-positive bacteria were consistently resistant to ceftazidime.

CONCLUSIONS AND CLINICAL RELEVANCE

Aerobic bacterial culture and antimicrobial susceptibility results for reptilian samples in this population indicated that aminoglycosides and trimethoprim-sulfamethoxazole or ampicillin and doxycycline could be considered as options for the empirical treatment of reptiles with infections caused by gram-negative or gram-positive bacteria, respectively.

Abstract

OBJECTIVE

To identify antimicrobial susceptibility patterns for aerobic bacteria isolated from reptilian samples and, from those patterns, identify antimicrobials that could be considered for empirical treatment of reptiles with suspected bacterial infections.

SAMPLES

129 bacterial isolates from 61 of 127 samples from 96 reptiles.

PROCEDURES

Medical records of reptiles (chelonian, crocodilian, lizard, and snake) presented to the zoological medical service of a veterinary teaching hospital between January 2005 and December 2016 were reviewed for submissions of patient samples for aerobic bacterial culture and susceptibility testing. Sample type, presence or absence of bacterial growth, and antimicrobial susceptibilities of isolated bacteria were recorded. The isolation frequency and the antimicrobial susceptibilities of bacterial genera and species were tabulated.

RESULTS

Pseudomonas spp and Enterococcus spp were the most frequently isolated gram-negative and gram-positive bacteria, respectively. Isolates of gram-negative bacteria frequently had susceptibility to amikacin (86%), gentamicin (95%), tobramycin (92%), and trimethoprim-sulfamethoxazole (83%), and gram-positive bacteria frequently had susceptibility to ampicillin (83%), chloramphenicol (92%), doxycycline (100%), and gentamicin (100%). Isolates of gram-positive bacteria were consistently resistant to ceftazidime.

CONCLUSIONS AND CLINICAL RELEVANCE

Aerobic bacterial culture and antimicrobial susceptibility results for reptilian samples in this population indicated that aminoglycosides and trimethoprim-sulfamethoxazole or ampicillin and doxycycline could be considered as options for the empirical treatment of reptiles with infections caused by gram-negative or gram-positive bacteria, respectively.

Reptiles have become increasingly popular as pets in recent decades in Europe and the United States,1,2 and therefore, they are presented more frequently to veterinarians because of various medical conditions, including those with bacterial causes.3 Veterinarians commonly administer antimicrobials to reptiles without the benefit of results of bacterial culture and antimicrobial susceptibility testing, sometimes because of the patient's need of immediate treatment and the pet owner's inability to afford the cost of testing. Yet sometimes the selected antimicrobial is not appropriate for the bacterial species causing the infection, or an antimicrobial is unnecessary because the patient does not have a bacterial infection. Therefore, veterinarians may unwittingly contribute to the development of bacterial resistance to antimicrobials, to the delay of an accurate diagnosis, and to an increased risk of precipitating an adverse event or toxicosis.4 Selection of an appropriate antimicrobial is also important so that the resident gastrointestinal microbiota of reptiles, which aid in optimal digestion and immune function, are maintained or minimally disrupted.5

Confirmation of a bacterial infection requires the isolation of bacteria from a biological sample via bacterial culture and a host response to the bacteria determined via cytologic or histologic examination. Furthermore, antimicrobial susceptibility testing is necessary to ensure selection of an appropriate antimicrobial. However, immediate determination of whether the sample contains gram-positive or gram-negative bacteria (by heat fixation of the sample on a microscope slide, Gram staining, and microscopic examination) may help guide veterinarians in choosing an antimicrobial while culture and susceptibility results are pending.

The objectives of the study reported here were to determine the most common bacteria isolated from reptilian samples submitted for aerobic bacterial culture and the antimicrobial susceptibility patterns of the isolates. We anticipated that antimicrobial susceptibilities would vastly differ between gram-negative and gram-positive bacteria, and antimicrobials commonly prescribed on the basis of empirical evidence (eg, potentiated sulfonamides and first-generation penicillins) would generally be ineffective, thereby necessitating the prescription of antimicrobials with a broader spectrum of antimicrobial activity (eg, fluoroquinolones and third- and fourth-generation cephalosporins).

Materials and Methods

Medical records review

Medical records of reptiles (chelonian, crocodilian, lizard, and snake species) presented to the zoological medical service of a veterinary teaching hospital between January 2005 and December 2016 were reviewed for submissions of patient samples for aerobic bacterial culture and susceptibility testing. Sample type, presence or absence of bacterial growth, and antimicrobial susceptibilities of isolated bacteria were recorded. Excluded from this study were results of culture and susceptibility tests for fecal samples and results of culture without concurrent antimicrobial susceptibilities. Results were also excluded for samples collected from the same site and patient more than once < 3 months from the date of the first submission.

Bacteriologic methods

Samples, including fresh tissue, fluids, and swab specimens,a submitted for aerobic bacterial culture were processed on the day of collection except when samples were collected outside of hours of laboratory operation. These samples were refrigerated at 4°C until they could be processed on the next business day. Samples were processed according to standard operating procedures of the laboratory. The laboratory was accredited by the American Association of Veterinary Laboratory Diagnosticians. The methodology of aerobic bacterial culture varied, depending on the type of sample and the part of the body from which it was obtained.6 Growth media inoculated with a sample were incubated for 48 hours at 30°C at ambient atmospheric pressure. No bacterial growth observed at 48 hours was deemed a negative result. Through 2014, bacterial isolates were identified via standard biochemical reactions7–9; after 2014, isolates were identified via an automated microbial identification system involving matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.b Bacteria that could not be identified with this system were considered to be environmental contaminants. Antimicrobial susceptibilities were determined by use of the disk diffusion method. Antimicrobial breakpoints, as defined in the guidelines of the Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing,10–31 were applied for all evaluated antimicrobials.

Data analysis

Data acquired from the medical records were reviewed, entered into an electronic spreadsheet,c and tabulated. Descriptive statistics were used to report the frequencies of sampled sites and bacterial genera and species and the results of Gram staining and antimicrobial susceptibility testing. The data for samples collected from reptiles that had been treated with antimicrobials within 14 days before sample collection were segregated from the data for samples collected from the remaining population, and results of culture (positive or negative) were compared between the 2 groups with the χ2 test. Statistical analysis was performed by use of commercially available software.d Values of P < 0.05 were considered significant.

Results

Between 2005 and 2016, 131 samples originating from 9 different body sites of 100 reptiles (40 lizards, 30 chelonians, 29 snakes, and 1 crocodilian) were submitted for aerobic bacterial culture and susceptibility testing. Two samples were excluded because they were of feces, and 2 other samples (1 cornea and 1 external [skin] lesion) were excluded because they were second submissions from the same site and patient (2 lizards) < 3 months from the date of the first submission. Of the remaining 127 samples, 61 samples (21 lizards, 26 chelonians, and 14 snakes; 48%) yielded bacterial growth for a total of 129 isolates. These 61 samples were most frequently obtained from external (skin and shell) lesions or abscesses (n = 21 [34%]), the oral cavity (11 [18%]), the cornea (10 [16%]), and the respiratory tract (8 [13%]), and these sites yielded growth of the greatest numbers of the bacterial isolates (external lesion or abscess, 20/129 [16%]; cornea, 13 [10%]; oral cavity, 14 [11%]; and respiratory tract, 11 [9%]). Other less frequently sampled sites contributed low numbers of bacterial isolates for testing (coelomic cavity, n = 6 [isolates, 7]; blood, 2 [4]; bone, 1 [1]; and unknown, 2 [3]).

The majority of bacterial isolates were gram negative (96/129 [74%]; Table 1). Thirty-four genera of bacteria were identified, and the genera of the most frequently isolated bacteria were Pseudomonas (n = 19), Enterococcus (15), Morganella (9), Staphylococcus (7), and Escherichia (7). The genera of less commonly isolated bacteria were Corynebacterium (5), Proteus (5), Stenotrophomonas (5), Salmonella (4), Enterobacter (4), Providencia (4), Aeromonas (3), Chryseobacterium (3), Citrobacter (3), Klebsiella (3), Pasteurella (3), Acinetobacter (2), Moraxella (2), Streptococcus (2), and Actinomycete, Alcaligenes, Bordetella, Chryseomonas, Comamonas, Clostridium, Empedobacter (Flavobacterium), Ochrobactrum, Pantoea, Psychrobacter, Serratia, and Vibrio (1 each). Twelve bacterial isolates for which the genera could not be determined (gram-negative bacilli [n = 10]; gram-positive bacillus [1]; and gram-positive coccus [1]) were likely environmental contaminants. Pseudomonas spp were isolated from samples obtained from 6 sites, predominantly from an external lesion or abscess (n = 7) and the oral cavity (6). Enterococcus spp were isolated from samples obtained from 7 sites, including the coelomic cavity (n = 4), an external lesion or abscess (3), and blood (3). Morganella morganii was isolated from 4 sites, often from an external lesion or abscess (n = 4) and the oral cavity (3). Escherichia coli was isolated from 3 sites, primarily from an external lesion or abscess (n = 5). Staphylococcus spp were isolated from 3 sites, primarily from the cornea (n = 4).

Table 1—

Antimicrobial susceptibilities classified as percentages susceptible (S), resistant (R), and intermediate (I) of 129 isolates of gram-negative and gram-positive bacteria identified via aerobic culture of 61 samples from reptiles (chelonian, crocodilian, lizard, and snake) presented to the zoological medical service of a veterinary teaching hospital between January 2005 and December 2016.

 Gram-negative (n = 96)Gram-positive (n = 33) 
AntimicrobialNo. of isolatesS (%)R (%)I (%)No. of isolatesS (%)R (%)I (%)
Amikacin79861041644560
AMC70386012681812
Ampicillin50366221883170
Azithromycin221373149444411
Bacitracin1118820888130
Carbenicillin9672211
Cefazolin1233588967330
Cefotaxime12255817
Cefotetan10405010
Cefpodoxime2259365100100
Ceftazidime42811721101000
Ceftiofur24712541464297
Cephalothin5338602683170
Chloramphenicol3482612129208
Ciprofloxacin317419711361846
Clindamycin19010001712826
Doxycycline1968265610000
Enrofloxacin818210926352342
Erythromycin10010017413524
Florfenicol2259329118299
Gatifloxacin7861407711414
Gentamicin629550610000
Gentamicin (HLAS)1100001110000
Imipenem786140410000
Marbofloxacin110000250050
Neomycin19532621967330
Nitrofurantoin520800101000
Ofloxacin1995509563311
Orbifloxacin3253252220106030
Oxacillin101000633670
Penicillin2349602277230
Polymyxin B19792109562222
Rifampin580020
Tetracycline55732441979210
Ticarcillin6276195
Tobramycin719290967330
TMS70831161753416
Vancomycin310000110000

— = Not determined. AMC = Amoxicillin–clavulanic acid. HLAS = High-level aminoglycoside susceptibility. Not all bacterial isolates were evaluated for susceptibility to each antimicrobial. Because of rounding, percentages may not add to 100%.

Isolates of gram-negative bacteria were frequently susceptible to aminoglycosides (amikacin [68/79 {86%}], gentamicin [60/63 {95%}], and tobramycin [65/71 {92%}] but not neomycin [10/19 {53%}]), second-generation fluoroquinolones (ciprofloxacin [23/31 {74%}], enrofloxacin [66/81 {82%}], and ofloxacin [18/19 {95%}] but not orbifloxacin [17/32 {53%}]), an advanced β-lactam antimicrobial (imipenem [6/7 {86%}]), a third-generation cephalosporin (ceftazidime [34/42 {81%}]), and TMS (58/70 [83%]; Table 1). Gram-negative bacteria were frequently resistant to penicillin (22/23 [96%]), first- and second-generation cephalosporins (cephalothin [32/53 {60%}] and cefotetan [5/10 {50%}]), clindamycin (19/19 [100%]), and azithromycin (16/22 [73%]).

Specifically, Pseudomonas spp had similar susceptibility patterns to those of all gram-negative bacteria for aminoglycosides, the second-generation fluoroquinolones ciprofloxacin and ofloxacin, and ceftazidime (Table 2). Compared with the antimicrobial susceptibilities of all gram-negative bacteria, Pseudomonas spp were more often resistant to amoxicillin–clavulanic acid (6/7 [86%] vs 42/70 [60%]), TMS (4/6 [67%] vs 8/70 [11%]), and the second-generation fluoroquinolones enrofloxacin (4/16 [25%] vs 8/81 [10%]) and orbifloxacin (4/7 [57%] vs 8/32 [25%]).

Table 2—

Antimicrobial susceptibilities of 19 isolates of Pseudomonas spp obtained via bacterial culture of reptilian samples of Table 1.

AntimicrobialNo. of isolatesS (%)R (%)I (%)
Amikacin1610000
AMC*714860
Bacitracin301000
Carbenicillin8503813
Ceftazidime1410000
Ciprofloxacin129270
Enrofloxacin17382538
Gatifloxacin210000
Gentamicin1310000
Imipenem510000
Neomycin211362736
Ofloxacin1110000
Orbifloxacin7295714
Penicillin2501000
Polymyxin B1191100
Ticarcillin1258338
Tobramycin1610000
TMS26176717

Pseudomonas spp expected to have intrinsic resistance. Not all bacterial isolates were evaluated for susceptibility to each antimicrobial. Because of rounding, percentages may not add to 100%.

See Table 1 for remainder of key.

Isolates of M morganii and E coli had similar susceptibility patterns to those of all gram-negative bacteria and Pseudomonas spp for aminoglycosides (amikacin, gentamicin, and tobramycin; not evaluated for susceptibility to neomycin), fluoroquinolones, and ceftazidime, and to those of all gram-negative bacteria for TMS (Tables 3 and 4). Isolates of M morganii and E coli had the same high frequency of susceptibility to tetracycline and ticarcillin.

Table 3—

Antimicrobial susceptibilities of 9 isolates of Morganella morganii obtained via bacterial culture of the reptilian samples of Table 1.

AntimicrobialNo. of isolatesS (%)R (%)I (%)
Amikacin810000
AMC*701000
Ampicillin*7147114
Azithromycin*201000
Ceftazidime310000
Cephalothin*701000
Chloramphenicol580020
Doxycycline410000
Enrofloxacin810000
Florfenicol110000
Gatifloxacin110000
Gentamicin710000
Orbifloxacin367033
Tetracycline*786140
Ticarcillin710000
Tobramycin710000
TMS888130

Morganella morganii expected to have intrinsic resistance. Not all bacterial isolates were evaluated for susceptibility to each antimicrobial. Because of rounding, percentages may not add to 100%.

See Table 1 for remainder of key.

Table 4—

Antimicrobial susceptibilities of 7 isolates of Escherichia coli obtained via bacterial culture of the reptilian samples of Table 1.

AntimicrobialNo. of isolatesS (%)R (%)I (%)
Amikacin710000
AMC7147114
Ampicillin520800
Cefotaxime110000
Cefotetan110000
Cefpodoxime210000
Ceftazidime110000
Ceftiofur110000
Cephalothin7147114
Chloramphenicol410000
Doxycycline310000
Enrofloxacin710000
Gentamicin710000
Orbifloxacin110000
Tetracycline786140
Ticarcillin710000
Tobramycin610000
TMS610000
Vancomycin110000

Not all bacterial isolates were evaluated for susceptibility to each antimicrobial. Because of rounding, percentages may not add to 100%.

See Table 1 for remainder of key.

Many isolates of gram-positive bacteria were susceptible to bacitracin (7/8 [88%]), chloramphenicol (11/12 [92%]), first- and second-generation cephalosporins (cephalothin [5/6 {83%}] and cefazolin [6/9 {67%}]), gentamicin (17/17 [100%]), penicillin (17/22 [77%]), and tetracycline (15/19 [79%]) and were often resistant to amikacin (9/16 [56%]), third- and fourth-generation cephalosporins (ceftazidime [11/11 {100%}] and ceftiofur [4/14 {29%}]), clindamycin (14/17 [82%]), fluoroquinolones (enrofloxacin [6/26 {23%}], ofloxacin [3/9 {33%}], and orbifloxacin [12/20 {60%}]), and TMS (7/17 [41%]; Table 1). All (100%) isolates of gram-positive bacteria evaluated for susceptibility to doxycycline (n = 6), gentamicin (13), and imipenem (4) were susceptible.

Similarly, isolates of Enterococcus spp were often susceptible to chloramphenicol, gentamicin, and penicillin and were often resistant to amikacin, fluoroquinolones, ceftazidime, clindamycin, and TMS (Table 5). All isolates of Staphylococcus spp were often susceptible to many antimicrobials for which they were evaluated (Table 6).

Table 5—

Antimicrobial susceptibilities of 15 isolates of Enterococcus spp obtained via bacterial culture of the reptilian samples of Table 1.

AntimicrobialsNo. of isolatesS (%)R (%)I (%)
Amikacin*601000
AMC1587013
Ampicillin129280
Azithromycin*650500
Ceftazidime*601000
Chloramphenicol888013
Ciprofloxacin603367
Clindamycin*601000
Doxycycline410000
Enrofloxacin15132760
Erythromycin*12502525
Florfenicol683017
Gatifloxacin250050
Gentamicin (HLAS)*1010000
Imipenem110000
Neomycin*201000
Orbifloxacin1576727
Penicillin1384150
Tetracycline1370310
TMS*601000
Vancomycin110000

Enterococcus spp expected to have intrinsic resistance (including gentamicin but not gentamicin HLAS). Not all bacterial isolates were evaluated for susceptibility to each antimicrobial. Because of rounding, percentages may not add to 100%.

See Table 1 for remainder of key.

Table 6—

Antimicrobial susceptibilities of 7 isolates of Staphylococcus spp obtained via aerobic culture of the reptilian samples of Table 1.

AntimicrobialNo. of isolatesS (%)R (%)I (%)
Amikacin310000
AMC450500
Ampicillin*333670
Bacitracin310000
Cefazolin310000
Ceftiofur6503317
Cephalothin475250
Chloramphenicol310000
Clindamycin4255025
Doxycycline110000
Enrofloxacin475250
Erythromycin201000
Fusidic acid110000
Gatifloxacin310000
Gentamicin410000
Marbofloxacin110000
Neomycin310000
Ofloxacin310000
Oxacillin450500
Penicillin367330
Polymyxin B*310000
Rifampicin210000
Tetracycline410000
Tobramycin310000
TMS410000

Staphylococcus spp expected to have intrinsic resistance. Not all bacterial isolates were evaluated for susceptibility to each antimicrobial. Because of rounding, percentages may not add to 100%.

See Table 1 for remainder of key.

Nine of the 61 (15%) samples yielding 22 (17%) bacterial isolates were collected from reptiles that had received topical or systemic antimicrobial treatment within the previous 14 days. No significant (P = 0.857) difference was identified between the treated group (9/18 [50%]) and the untreated group (52/109 [48%]) in the proportion of samples that yielded bacterial growth. Eight of 22 (36%) isolates from 4 samples were resistant to the antimicrobials that had been administered before culture and susceptibility results were available. Two isolates of Pseudomonas spp and 1 isolate of M morganii collected from the oral cavity were resistant to azithromycin. One isolate of Stenotrophomonas maltophilia collected from the nasal cavity was resistant to ciprofloxacin. One isolate each of Enterococcus spp and a gram-positive bacillus (most likely an environmental contaminant) collected from an external lesion or abscess was resistant to ceftazidime. One isolate each of Streptococcus sobrinus and Pseudomonas spp collected from the oral cavity was resistant to enrofloxacin.

Discussion

The findings of the present study highlighted antimicrobial susceptibilities for gram-negative and gram-positive bacteria isolated from aerobic cultures of samples collected from reptiles presented to the zoological medicine service at a veterinary teaching hospital. The number of studies and reviews1,3,4 on the use of antimicrobials in reptiles is limited, and consensus on the most appropriate empirical antimicrobial treatment is lacking.5 An appropriate antimicrobial stewardship program is important to minimize the development of antimicrobial resistance. To our knowledge, the present study was the first to characterize the antimicrobial susceptibilities of bacteria isolated from reptilian samples in the United States.

Most submitted samples (52%) did not yield bacterial growth within 48 hours after inoculation of growth media. Factors that may have contributed to the lack of bacterial growth despite the presence of viable bacteria and infection included sample technique and handling and fastidious bacteria. However, another consideration is the absence of a bacterial infection.

Gram-negative bacteria (74%) were more commonly identified than gram-positive bacteria (26%), a finding similar to that reported3 for bacteria isolated from reptilian samples in the United Kingdom. In the present study, gram-negative bacterial isolates, including all isolates of 3 of the 5 most frequently isolated bacteria—Pseudomonas spp, M morganii, and E coli—were susceptible to the aminoglycosides amikacin, gentamicin, and tobramycin. Therefore, aminoglycosides remain important for the treatment of gram-negative bacterial infections despite the higher risk of kidney injury with aminoglycosides, compared with the risk of other antimicrobials.32–34 Gentamicin may have a narrower margin of safety, and it has been reported to be more nephrotoxic than amikacin34,35; therefore, the use of amikacin or tobramycin as empirical treatment is likely a safer option. In lieu of an aminoglycoside, however, TMS may be a safer, equally effective option for infections caused by some gram-negative bacteria, including M morganii and E coli, with 83% of the gram-negative bacterial isolates in the present study susceptible to TMS. However, Pseudomonas spp, the most frequently isolated gram-negative bacteria of the present study, were often resistant (4/6 isolates) to TMS, reflecting the intrinsic resistance of the genus to this antimicrobial.36 Therefore, TMS is not recommended for treatment of infections caused by Pseudomonas spp. Salmonella spp were infrequently (3%) isolated, yet confirmation of an infection with these bacteria is important because of the risk of zoonosis.1,2 We identified that all 4 isolates (sample site: blood [1 isolate], external lesion [1], and unknown [2]) were susceptible to TMS and enrofloxacin.

Enterococcus spp were the most frequently isolated gram-positive bacteria. Like Pseudomonas spp, Enterococcus spp are inherently resistant to TMS,36 and all 6 isolates of Enterococcus spp that had been evaluated for susceptibility to TMS were resistant to TMS in the present study. However, unlike Pseudomonas spp, Enterococcus spp are also inherently resistant to aminoglycosides,36 and all isolates that were evaluated for susceptibility to amikacin and neomycin were resistant in the present study. Ten isolates were susceptible to high concentrations of gentamicin (high-level aminoglycoside susceptibility), but we cannot recommend gentamicin for the treatment of infections caused by Enterococcus spp because its in vitro susceptibility to high concentrations of gentamicin may not be applicable to situations in which gentamicin is administered as monotherapy. Alternatives for the treatment of infections caused by Enterococcus spp and other gram-positive bacteria may be doxycycline and penicillin. Although isolates of gram-positive bacteria were also frequently susceptible to chloramphenicol, this antimicrobial is not recommended because it is bacteriostatic.4 A few isolates of gram-positive bacteria that had been evaluated for susceptibility to imipenem were 100% susceptible, but the use of imipenem should be reserved because it is an important antimicrobial for the treatment of infections in humans caused by multidrug-resistant bacteria.37–40

Overall, many isolates of gram-negative bacteria were susceptible to ceftazidime (81%), and all 14 isolates of Pseudomonas spp plus all isolates of M morganii (3) and E coli (1) that were specifically evaluated for susceptibility to ceftazidime were susceptible, as expected. Also, isolates of gram-negative bacteria had susceptibility to ceftazidime that was comparable to that of other antimicrobials, including TMS. Surprisingly, however, all 11 gram-positive isolates that were evaluated for susceptibility to ceftazidime were resistant. Although ceftazidime is commonly administered to reptiles,41,42 possibly owing to its broad spectrum of antimicrobial activity and long half-life, we question its routine use, especially for the treatment of infections caused by gram-positive bacteria without supportive culture and antimicrobial susceptibility results.

In the present study, samples were obtained from reptiles that were and were not administered an antimicrobial, reflecting the reality of clinical veterinary practice in which samples may not be collected for aerobic bacterial culture and antimicrobial susceptibility testing until after treatment failure with administration of a previously prescribed antimicrobial. The proportion of samples collected from the treated reptiles that yielded bacterial growth did not significantly differ from the proportion of samples collected from untreated reptiles that yielded bacterial growth. Yet 8 bacterial isolates from 4 samples were resistant to the antimicrobials that had been administered before culture and susceptibility results were available. Therefore, the administered antimicrobial was not the appropriate choice, or if an infection was caused by multiple bacterial species, it may have been efficacious for inhibiting the growth of all except 1 bacterial species. We excluded the results of culture and susceptibility testing for additional samples that had been collected from the same site and patient < 3 months after submission of the first sample to avoid selection bias of bacterial isolates and their corresponding susceptibility patterns, but we included the results of testing for additional samples from the same site and patient if they had been collected ≥ 3 months after submission of the first sample. However, the percentage of multiple submissions from the same patient was low (6% [8/127]), and inclusion of these results likely did not skew our results.

The most important limitation of the present study was its retrospective nature. The antimicrobial-impregnated disks used for disk diffusion susceptibility testing varied throughout the study because of yearly updated recommended methods.22,23 Some bacterial isolates were inconsistently evaluated for susceptibilities to some antimicrobials, which impacted the interpretation of the overall in vitro susceptibilities to those antimicrobials. Likewise, samples frequently yielded growth of bacteria of many different genera such that 26 genera were represented by < 5 isolates, and 3 genera of the most frequent bacterial isolates were represented by < 10 isolates. Additionally, results from this study may not be applicable for the current reptilian population in the United States because results are from a single institution and represent an 11-year period. Therefore, results of our study should be interpreted cautiously.

Medical records did not include the results of Gram staining, and therefore, the correlation, if any, between those results and the bacterial species isolated via culture could not be determined. Prior to choosing an antimicrobial for the treatment of a suspected bacterial infection and while results of culture and susceptibility testing are pending or if the pet owner declines the submission of the sample for culture and susceptibility testing, determining whether the bacteria is gram-negative or gram-positive may help veterinarians with choosing an appropriate antimicrobial. Published data for reptiles are lacking for the percentage of agreement between the results of Gram staining and the bacterial species isolated via culture. Agreement between Gram stain results and bacterial species isolated via culture is only fair with samples collected from Amazon parrots43; despite this, we still recommend the Gram stain technique because it is easy and rapid for the detection and differentiation of gram-positive and gram-negative bacteria in clinical practice. For example, empirical selection of an antimicrobial for a patient with a suspected bacterial infection should be guided by the result of Gram staining plus knowledge of the common pathogens of the affected site and the antimicrobial's spectrum of activity, adverse effects, pharmacokinetics, and route of administration.44,45 Antimicrobials with a narrow spectrum of activity should be considered first because their administration may limit any negative effect on the resident gastrointestinal microbiota and the development of resistant bacteria.44,45

Future investigations are warranted, including of the trends of antimicrobial susceptibility of bacteria isolated from reptilian samples across multiple study sites within the United States. Such investigations would require standardization of the antimicrobial-impregnated disks used for susceptibility testing. Switching to the use of minimum inhibitory concentrations to determine antimicrobial susceptibility is also recommended. In addition, further investigation is needed of the clinical responses to antimicrobials administered on the basis of culture and susceptibility test results because in vitro susceptibility may not always correlate with clinical improvement.

Acknowledgments

No external funding was used in this study. The authors declare that there were no conflicts of interest.

Presented in abstract form at ExoticsCon, Atlanta, September 2018.

ABBREVIATIONS

TMS

Trimethoprim-sulfamethoxazole

Footnotes

a.

BBL culture swab, Becton Dickinson, Franklin Lakes, NJ.

b.

Vitek MS, BioMérieux, Durham, NC.

c.

Excel, version 15.38 for Mac, Microsoft Corp, Redmond, Wash.

d.

SPSS, version 24, IBM Corp, Armonk, NY.

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  • 38. Benčić I, Benčić I, Vukičević-Baudoin D. Imipenem consumption and gram-negative pathogen resistance to imipenem at Sestre Milosrdnice University Hospital. Acta Clin Croat 2001;40:185189.

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  • 39. Kahne D, Leimkuhler C, Lu W, et al. Glycopeptide and lipoglycopeptide antibiotics. Chem Rev 2005;105:425448.

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  • 41. Gibbons PM. Therapeutics. In: Mader DR, Divers SJ, eds. Current therapy in reptile medicine and surgery. St Louis: Elsevier Saunders, 2013;5769.

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  • 42. Klein NC, Cunha BA. Third-generation cephalosporins. Med Clin North Am 1995;79:705719.

  • 43. Evans EE, Mitchell MA, Whittington JK, et al. Measuring the level of agreement between cloacal Gram's stains and bacterial cultures in Hispaniolan Amazon parrots (Amazona ventralis). J Avian Med Surg 2014;28:290296.

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  • 44. Gibbons PM. Advances in reptile clinical therapeutics. J Exot Pet Med 2014;23:2138.

  • 45. Mitchell MA. Therapeutics. In: Mader DR, ed. Reptile medicine and surgery. 2nd ed. St Louis: Elsevier, 2006;631664.

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  • 10. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing. CLSI supplement M100. 26th ed. Wayne, Pa: CLSI, 2016.

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    • Export Citation
  • 11. CLSI. Performance standards for antimicrobial susceptibility testing. CLSI supplement M100. 25th ed. Wayne, Pa: CLSI, 2015.

  • 12. CLSI. Performance standards for antimicrobial susceptibility testing. CLSI supplement M100. 24th ed. Wayne, Pa: CLSI, 2014.

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  • 22. CLSI. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. CLSI standard VET01. 4th ed. Wayne, Pa: CLSI, 2013.

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    • Export Citation
  • 23. CLSI. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. CLSI standard VET01. 3rd ed. Wayne, Pa: CLSI, 2008.

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    • Export Citation
  • 24. CLSI. Development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobial agents. CLSI document VET02–A3. 2nd ed. Wayne, Pa: CLSI, 2002.

    • Search Google Scholar
    • Export Citation
  • 25. CLSI. Development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobial agent. CLSI document VET02–A3. 3rd ed. Wayne, Pa: CLSI, 2008.

    • Search Google Scholar
    • Export Citation
  • 26. The European Committee on Antimicrobial Susceptibility Testing. Clinical breakpoints – bacteria v 6.0. www.eucast.org/ast_of_bacteria/previous_versions_of_documents/. Accessed on Apr 13, 2019.

    • Search Google Scholar
    • Export Citation
  • 27. The European Committee on Antimicrobial Susceptibility Testing. Clinical breakpoints – bacteria v 5.0. www.eucast.org/ast_of_bacteria/previous_versions_of_documents/. Accessed on Apr 13, 2019.

    • Search Google Scholar
    • Export Citation
  • 28. The European Committee on Antimicrobial Susceptibility Testing. Clinical breakpoints – bacteria v 4.0. www.eucast.org/ast_of_bacteria/previous_versions_of_documents/. Accessed on Apr 13, 2019.

    • Search Google Scholar
    • Export Citation
  • 29. The European Committee on Antimicrobial Susceptibility Testing. Clinical breakpoints – bacteria v 3.1. www.eucast.org/ast_of_bacteria/previous_versions_of_documents/. Accessed on Apr 13, 2019.

    • Search Google Scholar
    • Export Citation
  • 30. The European Committee on Antimicrobial Susceptibility Testing. Clinical breakpoints – bacteria v 2.0. www.eucast.org/ast_of_bacteria/previous_versions_of_documents/. Accessed on Apr 13, 2019.

    • Search Google Scholar
    • Export Citation
  • 31. The European Committee on Antimicrobial Susceptibility Testing. Clinical breakpoints – bacteria v 1.3. www.eucast.org/ast_of_bacteria/previous_versions_of_documents/. Accessed on Apr 13, 2019.

    • Search Google Scholar
    • Export Citation
  • 32. Kaloyanides GJ, Pastoriza-Munoz E. Aminoglycoside nephrotoxicity. Kidney Int 1980;18:571582.

  • 33. Decker B, Molitoris BA. Aminoglycoside-induced nephrotoxicity. In: McQueen CA, ed. Comprehensive toxicology. 2nd ed. St Louis: Elsevier, 2010;329346.

    • Search Google Scholar
    • Export Citation
  • 34. Luft FC, Bloch R, Sloan RS, et al. Comparative nephrotoxicity of aminoglycoside antibiotics in rats. J Infect Dis 1978;138:541545.

  • 35. Montali RJ, Bush M, Smeller JM. The pathology of nephrotoxicity of gentamicin in snakes: a model for reptilian gout. Vet Pathol 1979;16:108115.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Leclercq R, Cantón R, Brown DFJ, et al. EUCAST expert rules in antimicrobial susceptibility testing. Clin Microbiol Infect 2013;19:141160.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Goldstein EJ, Citron DM. Comparative in vitro activity of imipenem and 15 other antimicrobial agents against clinically important aerobic and anaerobic bacteria. Clin Ther 1988;10:487515.

    • Search Google Scholar
    • Export Citation
  • 38. Benčić I, Benčić I, Vukičević-Baudoin D. Imipenem consumption and gram-negative pathogen resistance to imipenem at Sestre Milosrdnice University Hospital. Acta Clin Croat 2001;40:185189.

    • Search Google Scholar
    • Export Citation
  • 39. Kahne D, Leimkuhler C, Lu W, et al. Glycopeptide and lipoglycopeptide antibiotics. Chem Rev 2005;105:425448.

  • 40. James RC, Pierce JG, Okano A, et al. Redesign of glycopeptide antibiotics: back to the future. ACS Chem Biol 2012;7:797804.

  • 41. Gibbons PM. Therapeutics. In: Mader DR, Divers SJ, eds. Current therapy in reptile medicine and surgery. St Louis: Elsevier Saunders, 2013;5769.

    • Search Google Scholar
    • Export Citation
  • 42. Klein NC, Cunha BA. Third-generation cephalosporins. Med Clin North Am 1995;79:705719.

  • 43. Evans EE, Mitchell MA, Whittington JK, et al. Measuring the level of agreement between cloacal Gram's stains and bacterial cultures in Hispaniolan Amazon parrots (Amazona ventralis). J Avian Med Surg 2014;28:290296.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Gibbons PM. Advances in reptile clinical therapeutics. J Exot Pet Med 2014;23:2138.

  • 45. Mitchell MA. Therapeutics. In: Mader DR, ed. Reptile medicine and surgery. 2nd ed. St Louis: Elsevier, 2006;631664.

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