a b s t r a c t

Backround: Infections are a common problem in cardiac arrest survivors. Antimicrobial drugs are often adminis- tered in routine care during treatment of patients with mild therapeutic hypothermia (MTH). Because there is to date no evidence for the pharmacodynamics of antimicrobial drugs under MTH conditions, we investigated the in vitro activity of common antimicrobials against clinically relevant bacterial pathogens.

Material and methods: Activities of antimicrobial drugs against clinically relevant bacterial pathogens were assessed in vitro by disk diffusion and broth microdilution assays at normothermic (37?C) and hypothermic (32?C) conditions.

Results: Seventy-three bacterial isolates were tested in disk diffusion and 15 in broth microdilution assays. Mean differences in zone diameters and minimal inhibitory concentration ratios were 0.6 mm (95% confidence interval, 0.3-0.9 mm) and 0.98 (95% confidence interval, 0.95-1.02), respectively, meeting predefined criteria for equiva- lence of in vitro antimicrobial activity.

Conclusions: The presented data provide reassuring evidence that the intrinsic activity of antimicrobials seems to be unaltered in MTH. However, further studies evaluating the pharmacokinetics including target site concentra- tions of the respective drugs and in vivo pharmacodynamics are necessary to complement our understanding of the appropriate use of antimicrobials in MTH.

(C) 2015

1. Introduction

Mild therapeutic hypothermia (MTH) has shown to improve neuro- logical outcome and survival after cardiac arrest, and it therefore be- came the standard of care. Despite conflicting evidence from a recent randomized controlled clinical trial, major Resuscitation guidelines rec- ommend cooling patients to a target core body temperature of 32?C to 34?C for 12 to 24 hours after resuscitation beside a Temperature control with 36?C [1-3].

A common problem after cardiac arrest is the occurrence of bacterial infections, particularly of the lower respiratory tract-most commonly resulting from aspiration due to unprotected airway and thorax com- pression during the process of resuscitation. Also, pulmonary contusion

? Support: This study was conducted independently of any support in the form of equipment, drugs, or grants.

* Corresponding author at: Department of Medicine I, Division of Infectious Diseases

and Tropical Medicine, Medical University of Vienna, Wahringergurtel 18-20, 1090 Vien- na. Tel.: +43 404004440; fax: +43 4040044180.

E-mail addresses: [email protected] (C. Wallmuller), [email protected] (B. Herold), [email protected] (F. Sterz), [email protected] (A. Makristathis), [email protected] (M. Ramharter).

during the cardiopulmonary resuscitation efforts, emergency airway ac- cess, and mechanical ventilation are factors that favor the risk for infec- tions [4,5]. Mild therapeutic hypothermia inhibits proinflammatory response through inhibition of leukocyte migration, phagocytosis, and decreased synthesis of proinflammatory enzymes that may lead to a higher risk of infections [6,7]. Unfortunately, there is evidence for effica- cy in the phase of MTH only for few drugs. Most enzyme-mediated reac- tions are temperature dependent, and it may therefore be hypothesized that rates of drug metabolism and drug disposition may be altered in the host during MTH. Most importantly, antimicrobial activity may be al- tered because of changes in metabolism of the bacterial pathogen under hypothermic conditions. However, to date, no data are available on the antimicrobial activity of drugs in the context of MTH. This knowl- edge may however be pivotal because we know from previous studies that early onset pneumonia often occurs within the first 3 days, which is well within the phase of MTH [4]. Because of the current lack of data, it is currently unknown whether the antimicrobial activity is al- tered in MTH as compared with normothermia.

We therefore performed an in vitro assessment to compare the anti- microbial activity of commonly used antimicrobial drugs in normother- mic and mild therapeutic hypothermic conditions against clinically relevant bacterial pathogens.

http://dx.doi.org/10.1016/j.ajem.2015.07.005

0735-6757/(C) 2015

1446 C. Wallmuller et al. / American Journal of Emergency Medicine 33 (2015) 1445-1448

1. Material and methods
   1. Study design and setting

Antimicrobial susceptibility testing was performed by disk diffusion and broth microdilution assays [8]. Firstly, disk diffusion assays were performed for common bacterial pathogens causing infections and es- pecially pneumonia in critical ill patients including Escherichia coli, Kleb- siella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, and Enterococcus faecalis. Tested antimicrobials (following national recommendations) are listed in Table 1. In a second step, we performed the broth microdilution method to investigate minimal inhibitory concentrations (MICs) of meropenem, ciprofloxacin, ceftriaxon, amoxicillin, and amox- icillin/clavulanic acid for selected gram-positive and Gram-negative pathogens (S aureus, E coli, and S pneumoniae). Tested microorganisms consisted of clinical isolates of the Division of Clinical Microbiology, General Hospital of Vienna, Austria, and type strains. All drugs for the microdilution assays were obtained from Sigma-Aldrich (St Louis, MO) and for the disk diffusion assay from Master Diagnostics (Bootle, Merseyside, UK).

• 1. Disk diffusion method

Antimicrobial susceptibility testing by disk diffusion was performed following the European Committee on Antimicrobial Susceptibility Test- ing (EUCAST) recommendations, a regulatory organization that pro- vides guidelines an protocols for testing, using Mueller-Hinton agar plates (MH2; bioMerieux) [9]. For S pneumoniae and H influenzae, a Mueller-Hinton fastidious agar (MHF; Oxold Deutschland GmbH) was used. Testing was performed under aerobic conditions both at 37?C and 32?C in parallel. Incubation time was 18 hours. Disks with E faecalis and glycopeptides were measured after 24 hours. S pneumoniae and H influenzae were grown in 4% to 6% CO₂.The diameters of inhibition zones were measured using a ruler following EUCAST recommenda- tions. A maximum of 3 disks were applied onto a single agar plate. For each species, 1 American Type Culture Collection culture strain and 5 to 16 clinical isolates were tested. All assays were performed in dupli- cate and were done at least at 2 independent occasions. Incubation pe- riods for the disk diffusion assay were 18 hours (24 hours for E faecalis with glycopeptides) at 32?C and 37?C and 36 hours at 32?C to allow for increased growth under hypothermic conditions, respectively.

• 1. Microdilution method

Standard microdilution assay following the International Standard Organization ISO/FDIS 20776-1 was used to determinate MIC to confirm results from disk diffusion assays. Susceptibility panels in 96-well mi- crotiter plates contained ascending concentrations of antimicrobial agents. Plates were incubated for 20 hours in parallel at 37?C and 32?C, respectively. All tests were performed in duplicate, and MIC is re- ported as arithmetic means. For each species, 7 isolates and 1 American

Type Culture Collection culture strain were used. All assays were per- formed under identical conditions. Incubation periods for broth microdilution assay were 20 hours at 32?C and 37?C, respectively.

• 1. Statistical analysis

Growth inhibition zone diameters and MICs were nonnormally dis- tributed and were therefore depicted in descriptive analysis as median and 25% and 75% quartiles. Comparison of data between the 2 temper- ature conditions were performed by calculation of differences in zone diameters and ratios of MICs using nonparametric tests (Wilcoxon rank sum test). Proof of equivalent antimicrobial activity was assumed if the 95% confidence interval (CI) of the comparison did not cross a predefined equivalence margin (+-2 mm difference in zone diameter and +-10% of MIC ratios). These arbitrary thresholds were predefined based on the assumption of such a difference being of limited clinical significance because this is within the range of Interobserver variability.

1. Results

A total of 73 bacterial isolates were assessed in disk diffusion assays. No differences in zone diameters were observed at 32?C when incubat- ed upon incubation periods recommended by EUCAST and 36 hours (data not shown). Therefore, only assay data incubating 18 hours at the 2 respective temperatures were used for further analysis.

All assays were performed in parallel for the 2 temperature levels, such as that all procedures were identical (use of same broth, antibiotic solutions, reading utensils) except for the incubation temperature. Inhibi- tion diameters were recorded (data not shown), and differences in inhi- bition zone diameters were calculated and are depicted in Table 2. Medians and quartiles indicated no difference in diameters of inhibition zones between 37?C and 32?C, respectively. When testing for equivalence of the antimicrobial activity at the 2 temperature conditions, the mean difference in zone diameters was 0.6 mm (95% CI, 0.3-0.9 mm), indicating proof of equivalence based on the predefined equivalence margin.

To further confirm results obtained from disk diffusion assays, the

broth microdilution assay was performed for accurate estimation of the MICs. Fifteen bacterial isolates were used for this confirmatory assay. For statistical comparison between the 2 temperature conditions, median concentration ratios of MICs were computed and quartiles of the MIC were calculated and are depicted in Table 3. No differences were observed as evidenced by the distribution of median concentra- tion ratios (Table 3). In statistical analysis for equivalence, mean ratios of MICs demonstrated equivalent antimicrobial activity based on the predefined criteria (mean ratio, 0.98; 95% CI, 0.95-1.02).

1. Discussion

Results of this pilot study indicate that antibacterial activity of antimicrobials commonly used in clinical practice is not altered by tem- perature conditions between 32?C and 37?C. Because criteria for equiv- alence of in vitro activity were met, these data implicate that the activity

Table 1

Panel of clinically relevant bacterial pathogens and antimicrobials assessed in the disk diffusion assay

Bacteria Antimicrobial drugs

S pneumoniae Oxacillin, moxifloxacin, vancomycin, clindamycin, erythromycin

S aureus Oxacillin, clindamycin, erythromycin, fusidic acid, cefotaxin, gentamycin, tetracyclin, tigecyclin, mupirocin, linezolid, rifampicin, ciprofloxacin, fosfomycin trimethroprim

E coli Gentmycin, ciprofloxacin, trimethoprim, cefuroxim, cefotaxim, cefepime, cefpodoxim, amoxicllin/clavulanic acid, ertapenem, meropenem, cefoxitin, cefalexin, mecillinam, amikacin, temocillin, aztreonam

K pneumoniae Gentamycin, ciprofloxacin, trimethoprim, cefuroxim, cefotaxim, cefepime, cefpodoxim, amoxicilin/calvulanic acid, ertapenem, meropenem, cefoxitin, cefalexin, mecillinam, amikacin, temocillin, aztreonam

P aeruginosa Gentamycin, cefepime, meropenem, amikacin, piperazillin/tazobactam, ceftazidime

H influenza Erythromycin, tetracyclin, ciprofloxacin, amoxicillin/clavulanic acid, ampcillin

E faecalis Linezolid, ampicillin, vancomycin, teicoplanin

C. Wallmuller et al. / American Journal of Emergency Medicine 33 (2015) 1445-1448 1447

of the tested antimicrobial agents is similar under normothermic and hypothermic conditions in vitro. To further corroborate our findings from the disk diffusion assay, we therefore performed the broth microdilution assay for a subset of the most clinically relevant bacterial pathogens. Importantly, these assays confirmed our previous results and showed no differences in the antimicrobial activity between the 2 temperature groups. There were no differences evident for any of the 32 tested antimicrobial agents. This consistent finding is reassuring ev- idence that current clinical practice of using standard antimicrobials for the treatment of infections is supported at least from an in vitro phar- macodynamic perspective.

Previous published studies mainly focused on variations of antimi- crobial activity under hyperthermic conditions. Mackowiak and Marling-Cason [10] found in 1983 a positive correlation between tem- perature and serum sucsceptibility. The difference was most striking when 33?C and 37?C were compared with 41?C. Regarding the effect of hypothermia on drug disposition, metabolism, and response, previ- ous studies have focused on anesthetics, sedatives, Neuromuscular blocking agents, and anticonvulsants. These data provide evidence that the therapeutic index of these drugs is altered during hypothermia [11,12]. Furthermore, in the phase of rewarming, there happens a change in the drug concentrations of sedatives and analgetics, as report- ed by Bjelland et al [13]. Regarding antibotics, controversial data have been published for gentamicin in hypoxic ischemic encephalopathy under MTH. Liu et al [14] reported no differences in serum concentra- tions in MTH, whereas Frymoyer et al [15] reported decreased gentami- cin clearance.

Bacterial infections-particularly lower respiratory tract infections— are a common clinical finding in patients with cardiac arrest. Based on data of a prospective cohort, we estimate that approximately 50% of the resuscitated patients subsequently develop clinical signs of infection with a preference for pneumonia [16]. Furthermore, in a more recent study by Woo et al [17], the importance of pneumonia in patients

with cardiac arrest receiving MTH is underlined. Importantly, to date, it remains controversial whether these clinical and biochemical signs are due to bacterial infections-providing evidence for the use of antibiotics-or whether these signs are caused by a “sepsis-like syn- drome” representing inflammation due to reperfusion injury [18]. There is evidence that the high proportion of clinical diagnoses of infec- tion was even more often evident in individuals treated with MTH than in normothermic patients [19,20]. This finding is further supported by one of the landmark studies for MTH, where a trend towards a higher rate of infections in the hypothermia group was reported [21]. Despite these data indicating a higher risk for clinical signs of infection following MTH, no increase in mortality has been observed, potentially because of the appropriate management of infections and the medical benefits of hypothermia.

Because of this high Incidence of infections, antibiotics are often used in routine clinical care of patients after cardiac arrest. It is important to treat infectious complications as soon as possible, reflected by a recent study by Davies et al [22] who showed that early antibiotic treatment may lead to a decrease in mortality for resuscitated patients. In addition, Mongardon et al [20] reported that early-onset pneumonia occurred after a median of 1 day after cardiac arrest and was much more common than late-onset pneumonia. Based on these data and the initiation of MTH mostly at the time of admission in hospital, we are therefore con- vinced that more evidence on the pharmacodynamics and pharmacoki- netics of antimicrobials under MTH conditions is needed to guide evidence-based clinical management of these patients. This is even more so because recommendations for the use of MTH are likely to in- clude further clinical conditions, such as stroke or other neurological diseases in the future [23,24]. Finally, acute myocardial infarction, representing a disease affecting millions of people all over the world, constitutes another indication currently under investigation for the ap- plication of MTH because of its proposed potential to reduce infarct size [25,26]. Based on this scenario, it becomes likely that the use of

Table 2

Differences in growth inhibition by antimicrobials in disk diffusion assays when incubated at 32?C and 37?C, respectively

Disk diffusion assay?	S pneumoniae	S aureus	E coli	K pneumoniae	P aeruginosa	H influenzae	E faecalis
Mecillinam	ND	ND	-1 (-1 to 2)	-1 (-3 to 0)	ND	ND	ND
Temocillin	ND	ND	0 (-1 to 0)	-1 (-2 to -1)	ND	ND	ND
Ampicillin	ND	ND	ND	ND	ND	0 (-1 to 0)	0 (-1 to 1)
Oxacillin	-1 (0 to -2)	1 (0-2)	ND	ND	ND	ND	ND
Piperacillin/tazobactam	ND	ND	ND	ND	-1 (-2 to 1)	ND	ND
Amoxicillin/clavulanic acid	ND	ND	0 (-1 to 0)	0 (-1 to 0)	ND	-1 (-1 to 0)	ND
Moxifloxacin	-2 (-1 to -2)	ND	ND	ND	ND	ND	ND
Vancomycin	2 (1-3)	ND	ND	ND	ND	ND	-1 (-1 to 0)
Teicoplanin	ND	ND	ND	ND	ND	ND	-1 (-2 to 0)
Clindamycin	-1 (0 to -2)	-1 (-2 to 0)	ND	ND	ND	ND	ND
Erythromycin	0 (-2 to 0)	-2 (-2 to -1)	ND	ND	ND	-1 (-1 to -1)	ND
Gentamycin	ND	0 (-1-0)	-1 (-2 to 2)	0 (-1 to 1)	0 (-1 to 0)	ND	ND
Amikacin	ND	ND	-1 (-2 to 0)	0 (0-1)	0 (-1 to 1)	ND	ND
Tetracyclin	ND	-1 (-2 to 0)	ND	ND	ND	0 (-2 to 0)	ND
Tigecyclin	ND	0 (-1 to 0)	ND	ND	ND	ND	ND
Mupirocin	ND	-1 (-2 to 0)	ND	ND	ND	ND	ND
Linezolid	ND	-3 (-3 to -2)	ND	ND	ND	ND	ND
Rifampicin	ND	-2 (-3 to -1)	ND	ND	ND	ND	ND
Fosfomycin	ND	3 (0-6)	ND	ND	ND	ND	ND
Trimethoprim	ND	-3 (-4 to -2)	-1 (-3 to 0)	0 (-3 to 0)	ND	ND	ND
Ciprofloxacin	ND	1 (0-2)	-1 (-4 to 0)	-3 (-3 to 0)	ND	0 (-1 to 0)	ND
Cefuroxim	ND	ND	-1 (-2 to 0)	-1 (-2 to 0)	ND	ND	ND
Cefotaxim	ND	ND	-3 (-3 to -1)	-1 (-2 to 0)	ND	ND	ND
Cefoxitin	ND	0 (-1 to 0)	2 (0-3)	-1 (-2 to 0)	ND	ND	ND
Ceftazidim	ND	ND	ND	ND	0 (-1 to 0)	ND	ND
Cefepime	ND	ND	-2,5 (-4 to -1)	0 (-4 to 0)	-1 (-1 to 1)	ND	ND
Cefpodoxim	ND	ND	-2 (-2 to 1)	-1 (-3 to 0)	ND	ND	ND
Cefalexin	ND	ND	-2 (-2 to 0)	0 (-1 to 0)	ND	ND	ND
Ertapenem	ND	ND	-3 (-3 to -1)	-2 (-3 to -1)	ND	ND	ND
Meropenem	ND	ND	-3 (-4 to -2)	-1 (-1 to -1)	-1 (-1 to -1)	ND	ND
Aztreonam	ND	ND	-4 (-5 to -2)	-1 (-3 to 0)	ND	ND	ND
Linezolid	ND	ND	ND	ND	ND	ND	-1 (-2 to -1)

Abbreviation: ND = not done, because not part of standard testing panel.

* Data depicted as median difference of zone diameter of growth inhibition between zone diameter at 32?C and zone diameter at 37?C incubation temperature (25% and 75% quantiles).

1448 C. Wallmuller et al. / American Journal of Emergency Medicine 33 (2015) 1445-1448

Table 3

Differences in growth inhibition by antimicrobials in broth microdilution assay when incubated at 32?C and 37?C, respectively

Broth microdilution assay?	Meropenem	Ciprofloxacin	Ceftriaxon	Amoxicillin	Amoxicillin/clavulanic acid
E coli	1 (1-1)	1 (0.7-1)	1 (1-1)	1 (1-1)	1 (0.5-1)
S aureus	1 (1-1)	1 (0.8-1)	1 (0.8-1)	1 (0.8-1)	1 (0.8-1)
S pneumoniae	1 (1-1)	1 (0.5-1)	1 (1-1)	1 (1-1)	1 (1-1)

* Data depicted as median ratio of MIC at 32?C and 37?C incubation temperature (25% and 75% quantiles).

antimicrobials in hypothermic patients will increase in the near future, emphasizing the need for further scientific evidence for the efficacy of antibiotic treatments under MTH conditions. Several methodological limitations associated with the disk diffusion assay are acknowledged in the context of this study. Besides potential observer bias due to un- blinded investigators performing the inoculation and manual reading of the assays, temperature-dependent differences in the diffusion of drugs within the agarose gel may also-at least in theory-serve as po- tential confounder. Importantly, in vitro evaluations of antimicrobial drugs are not directly applicable to the clinical setting. Important deter- minants of clinical outcome include pharmacokinetics, including target site drug concentrations and altered metabolism of antibiotics in hypo- thermic conditions, which require further clinical evaluation.

1. Conclusion

The presented data provide reassuring evidence that the intrinsic ac- tivity of antimicrobials seems to be unaltered in MTH; however, further studies evaluating the pharmacokinetics of antimicrobials including tar- get site concentrations of the respective drugs are necessary to comple- ment our understanding of the appropriate use of antimicrobials in MTH.

Acknowledgments

We are grateful for the support of the Landsteiner Gesellschaft and the Global Infectious Disease Control Association in the preparation of the manuscript.

References

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3. Field JM, Hazinski MF, Sayre MR, Chameides L, Schexnayder SM, Hemphill R, et al. Part 1: executive summary: 2010 American Heart Association guidelines for cardiopulmo- nary resuscitation and emergency cardiovascular care. Circulation 2010;122:640-56.
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9. European Society of Clinical Microbiology and Infectious Diseases. European Committee on Antimicrobial Susceptibility Testing. Antimicrobial susceptibility testing EUCAST disk diffusion method, version 3.0; 2013[Available at: http://www.eucast.org/antimicrobial_ susceptibility_testing/disk_diffusion_methodology. Accessed May 4, 2014].
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13. Bjelland TW, Klepstad P, Haugen BO, Nilsen T, Salvesen O, Dale O. Concentrations of remifentanil, propofol, fentanyl, and midazolam during rewarming from therapeutic hypothermia. Acta Anaesthesiol Scand 2014;58:709-15.
14. Liu X, Borooah M, Stone J, Chakkarapani E, Thoresen M. Serum gentamicin concen- trations inencephalopathic infants are not affected by therapeutic hypothermia. Pe- diatrics 2009;124:310-5.
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->

Activity of antimicrobial drugs against bacterial pathogens under mild hypothermic conditions?

Christian Wallmuller, MD ^a,b, Birger Herold, MD ^b,c, Fritz Sterz, MD ^a,

Athanasios Makristathis, PhD ^b, Michael Ramharter, MD ^c,d,?

^a Department of Emergency Medicine, Medical University of Vienna, Wahringer Gurtel 18-20, 1090 Vienna, Austria

^b Division of Clinical Microbiology, Department of Laboratory Medicine, Medical University of Vienna, Wahringer Gurtel 18-20, 1090 Vienna, Austria

^c Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Wahringer Gurtel 18-20, 1090 Vienna, Austria

^d Institute fur Tropenmedizin, Universitat of Tubingen, Wilhelmstrasse 27, 72074 Tubingen, Germany

a r t i c l e i n f o

Article history:

Received 11 June 2015

Received in revised form 3 July 2015

Accepted 3 July 2015

a b s t r a c t

Backround: Infections are a common problem in cardiac arrest survivors. Antimicrobial drugs are often adminis- tered in routine care during treatment of patients with mild therapeutic hypothermia (MTH). Because there is to date no evidence for the pharmacodynamics of antimicrobial drugs under MTH conditions, we investigated the in vitro activity of common antimicrobials against clinically relevant bacterial pathogens.

Material and methods: Activities of antimicrobial drugs against clinically relevant bacterial pathogens were assessed in vitro by disk diffusion and broth microdilution assays at normothermic (37?C) and hypothermic (32?C) conditions.

Results: Seventy-three bacterial isolates were tested in disk diffusion and 15 in broth microdilution assays. Mean differences in zone diameters and minimal inhibitory concentration ratios were 0.6 mm (95% confidence interval, 0.3-0.9 mm) and 0.98 (95% confidence interval, 0.95-1.02), respectively, meeting predefined criteria for equiva- lence of in vitro antimicrobial activity.

Conclusions: The presented data provide reassuring evidence that the intrinsic activity of antimicrobials seems to be unaltered in MTH. However, further studies evaluating the pharmacokinetics including target site concentra- tions of the respective drugs and in vivo pharmacodynamics are necessary to complement our understanding of the appropriate use of antimicrobials in MTH.

(C) 2015

1. Introduction

Mild therapeutic hypothermia (MTH) has shown to improve neuro- logical outcome and survival after cardiac arrest, and it therefore be- came the standard of care. Despite conflicting evidence from a recent randomized controlled clinical trial, major resuscitation guidelines rec- ommend cooling patients to a target core body temperature of 32?C to 34?C for 12 to 24 hours after resuscitation beside a temperature control with 36?C [1-3].

A common problem after cardiac arrest is the occurrence of bacterial infections, particularly of the lower respiratory tract-most commonly resulting from aspiration due to unprotected airway and thorax com- pression during the process of resuscitation. Also, pulmonary contusion

? Support: This study was conducted independently of any support in the form of equipment, drugs, or grants.

* Corresponding author at: Department of Medicine I, Division of Infectious Diseases

and Tropical Medicine, Medical University of Vienna, Wahringergurtel 18-20, 1090 Vien- na. Tel.: +43 404004440; fax: +43 4040044180.

E-mail addresses: [email protected] (C. Wallmuller), [email protected] (B. Herold), [email protected] (F. Sterz), [email protected] (A. Makristathis), [email protected] (M. Ramharter).

during the cardiopulmonary resuscitation efforts, emergency airway ac- cess, and mechanical ventilation are factors that favor the risk for infec- tions [4,5]. Mild therapeutic hypothermia inhibits proinflammatory response through inhibition of leukocyte migration, phagocytosis, and decreased synthesis of proinflammatory enzymes that may lead to a higher risk of infections [6,7]. Unfortunately, there is evidence for effica- cy in the phase of MTH only for few drugs. Most enzyme-mediated reac- tions are temperature dependent, and it may therefore be hypothesized that rates of drug metabolism and drug disposition may be altered in the host during MTH. Most importantly, antimicrobial activity may be al- tered because of changes in metabolism of the bacterial pathogen under hypothermic conditions. However, to date, no data are available on the antimicrobial activity of drugs in the context of MTH. This knowl- edge may however be pivotal because we know from previous studies that early onset pneumonia often occurs within the first 3 days, which is well within the phase of MTH [4]. Because of the current lack of data, it is currently unknown whether the antimicrobial activity is al- tered in MTH as compared with normothermia.

We therefore performed an in vitro assessment to compare the anti- microbial activity of commonly used antimicrobial drugs in normother- mic and mild therapeutic hypothermic conditions against clinically relevant bacterial pathogens.

http://dx.doi.org/10.1016/j.ajem.2015.07.005

0735-6757/(C) 2015

1446 C. Wallmuller et al. / American Journal of Emergency Medicine 33 (2015) 1445-1448

1. Material and methods
   1. Study design and setting

Antimicrobial susceptibility testing was performed by disk diffusion and broth microdilution assays [8]. Firstly, disk diffusion assays were performed for common bacterial pathogens causing infections and es- pecially pneumonia in critical ill patients including Escherichia coli, Kleb- siella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, and Enterococcus faecalis. Tested antimicrobials (following national recommendations) are listed in Table 1. In a second step, we performed the broth microdilution method to investigate minimal inhibitory concentrations (MICs) of meropenem, ciprofloxacin, ceftriaxon, amoxicillin, and amox- icillin/clavulanic acid for selected gram-positive and gram-negative pathogens (S aureus, E coli, and S pneumoniae). Tested microorganisms consisted of clinical isolates of the Division of Clinical Microbiology, General Hospital of Vienna, Austria, and type strains. All drugs for the microdilution assays were obtained from Sigma-Aldrich (St Louis, MO) and for the disk diffusion assay from Master Diagnostics (Bootle, Merseyside, UK).

• 1. Disk diffusion method

Antimicrobial susceptibility testing by disk diffusion was performed following the European Committee on Antimicrobial Susceptibility Test- ing (EUCAST) recommendations, a regulatory organization that pro- vides guidelines an protocols for testing, using Mueller-Hinton agar plates (MH2; bioMerieux) [9]. For S pneumoniae and H influenzae, a Mueller-Hinton fastidious agar (MHF; Oxold Deutschland GmbH) was used. Testing was performed under aerobic conditions both at 37?C and 32?C in parallel. Incubation time was 18 hours. Disks with E faecalis and glycopeptides were measured after 24 hours. S pneumoniae and H influenzae were grown in 4% to 6% CO₂.The diameters of inhibition zones were measured using a ruler following EUCAST recommenda- tions. A maximum of 3 disks were applied onto a single agar plate. For each species, 1 American Type Culture Collection culture strain and 5 to 16 clinical isolates were tested. All assays were performed in dupli- cate and were done at least at 2 independent occasions. Incubation pe- riods for the disk diffusion assay were 18 hours (24 hours for E faecalis with glycopeptides) at 32?C and 37?C and 36 hours at 32?C to allow for increased growth under hypothermic conditions, respectively.

• 1. Microdilution method

Standard microdilution assay following the International Standard Organization ISO/FDIS 20776-1 was used to determinate MIC to confirm results from disk diffusion assays. Susceptibility panels in 96-well mi- crotiter plates contained ascending concentrations of antimicrobial agents. Plates were incubated for 20 hours in parallel at 37?C and 32?C, respectively. All tests were performed in duplicate, and MIC is re- ported as arithmetic means. For each species, 7 isolates and 1 American

Type Culture Collection culture strain were used. All assays were per- formed under identical conditions. Incubation periods for broth microdilution assay were 20 hours at 32?C and 37?C, respectively.

• 1. Statistical analysis

Growth inhibition zone diameters and MICs were nonnormally dis- tributed and were therefore depicted in descriptive analysis as median and 25% and 75% quartiles. Comparison of data between the 2 temper- ature conditions were performed by calculation of differences in zone diameters and ratios of MICs using nonparametric tests (Wilcoxon rank sum test). Proof of equivalent antimicrobial activity was assumed if the 95% confidence interval (CI) of the comparison did not cross a predefined equivalence margin (+-2 mm difference in zone diameter and +-10% of MIC ratios). These arbitrary thresholds were predefined based on the assumption of such a difference being of limited clinical significance because this is within the range of interobserver variability.

1. Results

A total of 73 bacterial isolates were assessed in disk diffusion assays. No differences in zone diameters were observed at 32?C when incubat- ed upon incubation periods recommended by EUCAST and 36 hours (data not shown). Therefore, only assay data incubating 18 hours at the 2 respective temperatures were used for further analysis.

All assays were performed in parallel for the 2 temperature levels, such as that all procedures were identical (use of same broth, antibiotic solutions, reading utensils) except for the incubation temperature. Inhibi- tion diameters were recorded (data not shown), and differences in inhi- bition zone diameters were calculated and are depicted in Table 2. Medians and quartiles indicated no difference in diameters of inhibition zones between 37?C and 32?C, respectively. When testing for equivalence of the antimicrobial activity at the 2 temperature conditions, the mean difference in zone diameters was 0.6 mm (95% CI, 0.3-0.9 mm), indicating proof of equivalence based on the predefined equivalence margin.

To further confirm results obtained from disk diffusion assays, the

broth microdilution assay was performed for accurate estimation of the MICs. Fifteen bacterial isolates were used for this confirmatory assay. For statistical comparison between the 2 temperature conditions, median concentration ratios of MICs were computed and quartiles of the MIC were calculated and are depicted in Table 3. No differences were observed as evidenced by the distribution of median concentra- tion ratios (Table 3). In statistical analysis for equivalence, mean ratios of MICs demonstrated equivalent antimicrobial activity based on the predefined criteria (mean ratio, 0.98; 95% CI, 0.95-1.02).

1. Discussion

Results of this pilot study indicate that antibacterial activity of antimicrobials commonly used in clinical practice is not altered by tem- perature conditions between 32?C and 37?C. Because criteria for equiv- alence of in vitro activity were met, these data implicate that the activity

Table 1

Panel of clinically relevant bacterial pathogens and antimicrobials assessed in the disk diffusion assay

Bacteria Antimicrobial drugs

S pneumoniae Oxacillin, moxifloxacin, vancomycin, clindamycin, erythromycin

S aureus Oxacillin, clindamycin, erythromycin, fusidic acid, cefotaxin, gentamycin, tetracyclin, tigecyclin, mupirocin, linezolid, rifampicin, ciprofloxacin, fosfomycin trimethroprim

E coli Gentmycin, ciprofloxacin, trimethoprim, cefuroxim, cefotaxim, cefepime, cefpodoxim, amoxicllin/clavulanic acid, ertapenem, meropenem, cefoxitin, cefalexin, mecillinam, amikacin, temocillin, aztreonam

K pneumoniae Gentamycin, ciprofloxacin, trimethoprim, cefuroxim, cefotaxim, cefepime, cefpodoxim, amoxicilin/calvulanic acid, ertapenem, meropenem, cefoxitin, cefalexin, mecillinam, amikacin, temocillin, aztreonam

P aeruginosa Gentamycin, cefepime, meropenem, amikacin, piperazillin/tazobactam, ceftazidime

H influenza Erythromycin, tetracyclin, ciprofloxacin, amoxicillin/clavulanic acid, ampcillin

E faecalis Linezolid, ampicillin, vancomycin, teicoplanin

C. Wallmuller et al. / American Journal of Emergency Medicine 33 (2015) 1445-1448 1447

of the tested antimicrobial agents is similar under normothermic and hypothermic conditions in vitro. To further corroborate our findings from the disk diffusion assay, we therefore performed the broth microdilution assay for a subset of the most clinically relevant bacterial pathogens. Importantly, these assays confirmed our previous results and showed no differences in the antimicrobial activity between the 2 temperature groups. There were no differences evident for any of the 32 tested antimicrobial agents. This consistent finding is reassuring ev- idence that current clinical practice of using standard antimicrobials for the treatment of infections is supported at least from an in vitro phar- macodynamic perspective.

Previous published studies mainly focused on variations of antimi- crobial activity under hyperthermic conditions. Mackowiak and Marling-Cason [10] found in 1983 a positive correlation between tem- perature and serum sucsceptibility. The difference was most striking when 33?C and 37?C were compared with 41?C. Regarding the effect of hypothermia on drug disposition, metabolism, and response, previ- ous studies have focused on anesthetics, sedatives, Neuromuscular blocking agents, and anticonvulsants. These data provide evidence that the therapeutic index of these drugs is altered during hypothermia [11,12]. Furthermore, in the phase of rewarming, there happens a change in the drug concentrations of sedatives and analgetics, as report- ed by Bjelland et al [13]. Regarding antibotics, controversial data have been published for gentamicin in hypoxic ischemic encephalopathy under MTH. Liu et al [14] reported no differences in serum concentra- tions in MTH, whereas Frymoyer et al [15] reported decreased gentami- cin clearance.

Bacterial infections-particularly lower respiratory tract infections- are a common clinical finding in patients with cardiac arrest. Based on data of a prospective cohort, we estimate that approximately 50% of the resuscitated patients subsequently develop clinical signs of infection with a preference for pneumonia [16]. Furthermore, in a more recent study by Woo et al [17], the importance of pneumonia in patients

with cardiac arrest receiving MTH is underlined. Importantly, to date, it remains controversial whether these clinical and biochemical signs are due to bacterial infections-providing evidence for the use of antibiotics-or whether these signs are caused by a “sepsis-like syn- drome” representing inflammation due to reperfusion injury [18]. There is evidence that the high proportion of clinical diagnoses of infec- tion was even more often evident in individuals treated with MTH than in normothermic patients [19,20]. This finding is further supported by one of the landmark studies for MTH, where a trend towards a higher rate of infections in the hypothermia group was reported [21]. Despite these data indicating a higher risk for clinical signs of infection following MTH, no increase in mortality has been observed, potentially because of the appropriate management of infections and the medical benefits of hypothermia.

Because of this high incidence of infections, antibiotics are often used in routine clinical care of patients after cardiac arrest. It is important to treat infectious complications as soon as possible, reflected by a recent study by Davies et al [22] who showed that early antibiotic treatment may lead to a decrease in mortality for resuscitated patients. In addition, Mongardon et al [20] reported that early-onset pneumonia occurred after a median of 1 day after cardiac arrest and was much more common than late-onset pneumonia. Based on these data and the initiation of MTH mostly at the time of admission in hospital, we are therefore con- vinced that more evidence on the pharmacodynamics and pharmacoki- netics of antimicrobials under MTH conditions is needed to guide evidence-based clinical management of these patients. This is even more so because recommendations for the use of MTH are likely to in- clude further clinical conditions, such as stroke or other neurological diseases in the future [23,24]. Finally, acute myocardial infarction, representing a disease affecting millions of people all over the world, constitutes another indication currently under investigation for the ap- plication of MTH because of its proposed potential to reduce infarct size [25,26]. Based on this scenario, it becomes likely that the use of

Table 2

Differences in growth inhibition by antimicrobials in disk diffusion assays when incubated at 32?C and 37?C, respectively

Disk diffusion assay?	S pneumoniae	S aureus	E coli	K pneumoniae	P aeruginosa	H influenzae	E faecalis
Mecillinam	ND	ND	-1 (-1 to 2)	-1 (-3 to 0)	ND	ND	ND
Temocillin	ND	ND	0 (-1 to 0)	-1 (-2 to -1)	ND	ND	ND
Ampicillin	ND	ND	ND	ND	ND	0 (-1 to 0)	0 (-1 to 1)
Oxacillin	-1 (0 to -2)	1 (0-2)	ND	ND	ND	ND	ND
Piperacillin/tazobactam	ND	ND	ND	ND	-1 (-2 to 1)	ND	ND
Amoxicillin/clavulanic acid	ND	ND	0 (-1 to 0)	0 (-1 to 0)	ND	-1 (-1 to 0)	ND
Moxifloxacin	-2 (-1 to -2)	ND	ND	ND	ND	ND	ND
Vancomycin	2 (1-3)	ND	ND	ND	ND	ND	-1 (-1 to 0)
Teicoplanin	ND	ND	ND	ND	ND	ND	-1 (-2 to 0)
Clindamycin	-1 (0 to -2)	-1 (-2 to 0)	ND	ND	ND	ND	ND
Erythromycin	0 (-2 to 0)	-2 (-2 to -1)	ND	ND	ND	-1 (-1 to -1)	ND
Gentamycin	ND	0 (-1-0)	-1 (-2 to 2)	0 (-1 to 1)	0 (-1 to 0)	ND	ND
Amikacin	ND	ND	-1 (-2 to 0)	0 (0-1)	0 (-1 to 1)	ND	ND
Tetracyclin	ND	-1 (-2 to 0)	ND	ND	ND	0 (-2 to 0)	ND
Tigecyclin	ND	0 (-1 to 0)	ND	ND	ND	ND	ND
Mupirocin	ND	-1 (-2 to 0)	ND	ND	ND	ND	ND
Linezolid	ND	-3 (-3 to -2)	ND	ND	ND	ND	ND
Rifampicin	ND	-2 (-3 to -1)	ND	ND	ND	ND	ND
Fosfomycin	ND	3 (0-6)	ND	ND	ND	ND	ND
Trimethoprim	ND	-3 (-4 to -2)	-1 (-3 to 0)	0 (-3 to 0)	ND	ND	ND
Ciprofloxacin	ND	1 (0-2)	-1 (-4 to 0)	-3 (-3 to 0)	ND	0 (-1 to 0)	ND
Cefuroxim	ND	ND	-1 (-2 to 0)	-1 (-2 to 0)	ND	ND	ND
Cefotaxim	ND	ND	-3 (-3 to -1)	-1 (-2 to 0)	ND	ND	ND
Cefoxitin	ND	0 (-1 to 0)	2 (0-3)	-1 (-2 to 0)	ND	ND	ND
Ceftazidim	ND	ND	ND	ND	0 (-1 to 0)	ND	ND
Cefepime	ND	ND	-2,5 (-4 to -1)	0 (-4 to 0)	-1 (-1 to 1)	ND	ND
Cefpodoxim	ND	ND	-2 (-2 to 1)	-1 (-3 to 0)	ND	ND	ND
Cefalexin	ND	ND	-2 (-2 to 0)	0 (-1 to 0)	ND	ND	ND
Ertapenem	ND	ND	-3 (-3 to -1)	-2 (-3 to -1)	ND	ND	ND
Meropenem	ND	ND	-3 (-4 to -2)	-1 (-1 to -1)	-1 (-1 to -1)	ND	ND
Aztreonam	ND	ND	-4 (-5 to -2)	-1 (-3 to 0)	ND	ND	ND
Linezolid	ND	ND	ND	ND	ND	ND	-1 (-2 to -1)

Abbreviation: ND = not done, because not part of standard testing panel.

* Data depicted as median difference of zone diameter of growth inhibition between zone diameter at 32?C and zone diameter at 37?C incubation temperature (25% and 75% quantiles).

1448 C. Wallmuller et al. / American Journal of Emergency Medicine 33 (2015) 1445-1448

Table 3

Differences in growth inhibition by antimicrobials in broth microdilution assay when incubated at 32?C and 37?C, respectively

Broth microdilution assay?	Meropenem	Ciprofloxacin	Ceftriaxon	Amoxicillin	Amoxicillin/clavulanic acid
E coli	1 (1-1)	1 (0.7-1)	1 (1-1)	1 (1-1)	1 (0.5-1)
S aureus	1 (1-1)	1 (0.8-1)	1 (0.8-1)	1 (0.8-1)	1 (0.8-1)
S pneumoniae	1 (1-1)	1 (0.5-1)	1 (1-1)	1 (1-1)	1 (1-1)

* Data depicted as median ratio of MIC at 32?C and 37?C incubation temperature (25% and 75% quantiles).

antimicrobials in hypothermic patients will increase in the near future, emphasizing the need for further scientific evidence for the efficacy of antibiotic treatments under MTH conditions. Several methodological limitations associated with the disk diffusion assay are acknowledged in the context of this study. Besides potential observer bias due to un- blinded investigators performing the inoculation and manual reading of the assays, temperature-dependent differences in the diffusion of drugs within the agarose gel may also-at least in theory-serve as po- tential confounder. Importantly, in vitro evaluations of antimicrobial drugs are not directly applicable to the clinical setting. Important deter- minants of clinical outcome include pharmacokinetics, including target site drug concentrations and altered metabolism of antibiotics in hypo- thermic conditions, which require further clinical evaluation.

1. Conclusion

The presented data provide reassuring evidence that the intrinsic ac- tivity of antimicrobials seems to be unaltered in MTH; however, further studies evaluating the pharmacokinetics of antimicrobials including tar- get site concentrations of the respective drugs are necessary to comple- ment our understanding of the appropriate use of antimicrobials in MTH.

Acknowledgments

We are grateful for the support of the Landsteiner Gesellschaft and the Global Infectious Disease Control Association in the preparation of the manuscript.

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