Reasons for performing study
Sepsis is an important cause of death in foals. Knowledge of which pathogens are likely to be involved is important for selection of antimicrobial drugs for initial treatment.
Sepsis is an important cause of death in foals. Knowledge of which pathogens are likely to be involved is important for selection of antimicrobial drugs for initial treatment.
To identify temporal trends in prevalence of bacteria isolated from foals with sepsis between 1979 and 2010.
Retrospective review of medical records.
All foals ≤30 days of age presented to the Veterinary Medical Teaching Hospital (VMTH) at the University of California, Davis between 1979 and 2010, with a diagnosis of sepsis confirmed by culture of bacteria from blood or internal organs (antemortem or at necropsy), were included in the study. Conventional microbiological methods were used to identify isolated organisms. The Cochran–Armitage trend test was used for statistical analysis.
The percentage of Gram-positive isolates increased significantly over the years. The percentage Enterobacteriacea, and Klebsiella spp. in particular, decreased over time. Enterococcus spp. isolates were cultured more often in recent years.
Whereas Gram-negative bacteria, particularly Enterobacteriaceae, remain the most common isolates from neonatal foals with sepsis, the prevalence of Gram-positive bacteria is increasing. This trend underlines the importance of including antimicrobial drugs active against both Gram-positive and Gram-negative bacteria in treatment protocols while awaiting the results of bacteriological culture and susceptibility tests. The increased prevalence of Enterococcus spp. is of concern because antimicrobial susceptibility patterns for enterococci are unpredictable and enterococci can also act as donors of antimicrobial resistance genes to other bacteria.
Bacterial infection is a leading cause of death in foals during the first few weeks of life in North America . Signs of sepsis in the foal can initially be subtle and share features in common with other infectious and noninfectious conditions. Onset and nature of clinical signs, which frequently progress rapidly, are influenced by the pathogen involved, immune status of the foal and other factors [2, 3]. Infection can occur in utero or soon after birth via portals of entry that include the umbilicus, respiratory tract and wounds, although the gastrointestinal (GI) tract is the predominant portal of entry for infection [4-7]. Failure of passive transfer (FPT) of colostral antibody has long been recognised as a risk factor for the development of sepsis [8, 9]. Ingestion of adequate amounts of colostrum during the first few hours of life also plays an important nonimmunological role in preventing acquisition of infection by ‘closing the neonatal gut’ to translocation of macromolecules, including bacteria . Previous reports documenting that Gram-negative enteric bacteria are the predominant isolates from neonatal foals with sepsis, provide further evidence of the importance of the GI tract as a portal of entry for bacteria [2, 3, 5, 11-20].
Because signs of sepsis typically progress rapidly and frequently lead to death, aggressive early treatment with appropriate antimicrobials and diligent supportive care are necessary to successfully manage infected foals, while awaiting the results of culture and susceptibility tests performed on samples collected from the foal. Selection of antimicrobials for initial treatment should be based, at least in part, on historical information regarding the bacterial species most likely to be isolated from infected foals from a particular farm, facility or geographic location and the susceptibility of these isolates to particular antimicrobials, together with clinical indicators that may be associated with infection with specific groups or species of bacteria [2, 3, 5, 6, 11-20]. Because the antimicrobial activity of different classes of antimicrobial drugs may be predominantly or exclusively restricted to bacteria with specific Gram-staining characteristics, distinguishing foals infected with Gram-positive bacteria from those infected with Gram-negative bacteria would potentially be helpful in guiding selection of antimicrobials for initial treatment .
This study represents an extension of work that has been ongoing at the University of California (UC), Davis for many years, the overall objective of which is to generate quantitative antimicrobial susceptibility data to guide rational selection of antimicrobial drugs for inclusion in treatment protocols for foals with sepsis. The specific aim of the current study is to determine the prevalence of bacteria isolated from foals with sepsis and to evaluate potential temporal trends.
The case records of foals ≤30 days of age, presented to the William R. Pritchard Veterinary Medical Teaching Hospital (VMTH), University of California, Davis, USA, between January 1979 and December 2010, were reviewed. Data recorded in the medical record at admission and during hospitalisation of the foal were retrieved from the Veterinary Medical and Administrative Computing System (VMACS), as used by VMTH personnel at UC Davis, or from stored paper records if the data had not been entered into VMACS.
Records for those foals with a diagnosis of sepsis confirmed by culture of bacteria from blood or multiple internal organs were selected for further evaluation. Foals from which samples were collected by tracheal wash or by swabbing the umbilicus after surgical preparation, were also selected for further evaluation. Cases were included only if they showed clinical or laboratory signs consistent with systemic sepsis (fever [>38.9°C], neutropenia or neutrophilia [<4.0 × 109/l or >12.0 × 109/l], increased band neutrophil count [>0.05 × 109/l], toxic changes in neutrophils, fibrinogenaemia [>4.0 g/l], hypoglycaemia [<4.4 mmol/l], metabolic acidosis, scleral injection, petechiation, anterior uveitis, diarrhoea, respiratory distress or joint swelling). A total of 588 foals met the criteria for inclusion in the study.
Samples retrieved from foals with sepsis originated from several locations and 1–3 blood cultures were obtained per foal. Blood (5–10 ml) was aseptically collected from the jugular or cephalic vein or through jugular catheters at the time of placement using aseptic technique (often for first cultures, during recent years) for bacteriological culture, after removal of hair with a safety razor and preparation of the site with povidone-iodine scrub solution and alcohol. Culture of blood was performed using broth inoculation, with or without antibiotic resin (Trypticase Soy Broth)a or by the lysis-centrifugation method (Isolator)b.
Foals that died or were subjected to euthanasia were examined post mortem, during which samples from internal organs (e.g. liver, kidney, spleen, brain, body cavity or joint) were retrieved aseptically for bacteriological culture. All samples collected ante- or post mortem were submitted to the VMTH Microbiology Diagnostic Laboratory (MDL) at UC Davis for bacterial culture, identification and susceptibility testing. Conventional microbiological methods were used to identify isolated organisms.
When multiple isolates belonging to different bacterial species were cultured from one sample or from samples taken at different points in time or from different locations in a particular foal, all isolates were included in the present study. When multiple isolates of the same bacterial species were retrieved, they were considered to be the same isolate if their colony morphology, biochemical characteristics and antimicrobial susceptibility patterns were identical. Otherwise, isolates from different samples were considered to be different strains of the same bacterial species and both were included in the present study. Using the above criteria, a total of 1091 bacterial isolates from 588 foals were included.
Three time periods were established in order to identify potentially significant temporal trends in prevalence of bacterial isolates. Time periods were selected to take into account changes in approaches to antimicrobial use in neonatal foals at UC Davis during the 31 years of the study, as well as the desirability of including similar numbers of bacterial isolates in each time period. The time periods selected were 1979–1990, 1991–1997 and 1998–2010. Prior to 1990, gentamicin was the aminoglycoside antimicrobial of choice for inclusion in treatment regimens for sepsis in foals. A change in the approach to initial antimicrobial therapy was made in 1990 based on publication of a study documenting that a substantially higher proportion of Enterobacteriaceae isolated from foals with sepsis were susceptible to amikacin than to gentamicin . In 1997, an article was published advocating the prophylactic use of antimicrobials in foals that were born unobserved or had recognised risk factors . Subsequent to this publication, veterinarians at the VMTH and in the referral area increased their prophylactic use of antimicrobials in neonatal foals.
Changes in the specific antimicrobial drugs and dosing regimens used to treat foal with sepsis at the VMTH over the years are outlined below:
Penicillin G (20,000–40,000 iu/kg bwt i.v. q. 6 h) and kanamycin (5 mg/kg bwt i.m. q. 8 h) or trimethoprim/sulfamethoxazole (30 mg/kg bwt per os q. 12 h).
Late 1970s and 1980s:
Gentamicin (2.2 mg/kg bwt i.v. q. 8 h or 3.3 mg/kg bwt i.v. q. 12 h) and penicillin G (20,000–40,000 iu/kg bwt i.v. q. 6 h) or ampicillin (20 mg/kg bwt i.v. q. 6–8 h).
Amikacin (7 mg/kg bwt i.v. q. 8 h or 10 mg/kg bwt i.v. q. 12 h until 1995 and 21–25 mg/kg bwt i.v. q. 24 h from 1995 until the present) and ampicillin (20 mg/kg bwt i.v. q. 6-8 h), or ceftiofur sodium (5–10 mg/kg bwt i.v. or i.m. q. 12 h).
The Cochran–Armitage trend test was performed to determine temporal differences in prevalence of bacterial isolates between the 3 timeframes: 1979–1990; 1991–1997; 1998–2010. Analysis of this data was performed using commercial software (StatXact Version 9.0)c. Results were considered significant if the P value was ≤0.05.
A total of 1091 bacteria were isolated from 588 foals (Table 1). Three hundred and nine foals (52.6%) had a mixed infection. The mean number of isolates per foal was 1.85 and the median was 1 isolate. Seven hundred and sixty-six (70.2%) of the isolates were Gram-negative bacteria, whereas 325 (29.8%) were Gram-positive bacteria. Escherichia coli was the bacterial species most frequently isolated, accounting for 28.8% of all isolates. Other Gram-negative bacteria cultured included Actinobacillus spp. (13.9%), Klebsiella spp. (7.3%), Enterobacter spp. (3.8%), Salmonella spp. (2.9%), Pseudomonas spp. (2.5%) and Proteus spp. (2.2%). Gram-positive organisms included Streptococcus spp. (14.8%; 9.4% β-haemolytic streptococci and 5.3% other Streptococcus spp.), Enterococcus spp. (7.2%) and Staphylococcus spp. (5.3%). Other bacteria were cultured less frequently (<1.1%).
|Bacterial species||No. isolates||Percentage of total isolates (%)|
|Other Gram-negative isolates||64||5.9|
|Other Gram-positive isolates||27||2.5|
The numbers of isolates in each time period were as follows: 1979–1990: 328 isolates, 1991–1997: 415 isolates, 1998–2010: 348 isolates. Whereas E. coli, Actinobacillus spp. and Streptococcus spp. remained the most prevalent isolates in all time periods significant temporal changes were observed with regard to the prevalence of several bacterial species or groups of bacteria (Table 2).
|Bacterial species||1979–1990 (n = 328)||1991–1997 (n = 415)||1998–2010 (n = 348)|
|No. isolates||Percentage of total isolates (%)||No. isolates||Percentage of total isolates (%)||No. isolates||Percentage of total isolates (%)|
|Other Gram-negative isolates||16||4.9||22||5.3||26||7.5|
|Coagulase positive staphylococci||8||2.4||11||2.7||12||3.4|
|Coagulase negative staphylococci||6||1.8||10||2.4||8||2.3|
|Other Gram-positive isolates||6||1.8||11||2.7||10||2.9|
The percentage of Gram-positive isolates increased significantly over the years (1979–1990: 25.9%, 1991–1997: 27.7%, 1998–2010: 35.9%, P value 0.0042).
The percentage of Enterobacteriaceae decreased between the time periods 1991–1997 and 1998 and 2010 (1979–1990: 49.1%, 1991–1997: 52.5%, 1998–2010: 41.4%, P value 0.041). The relative frequency of isolation of Klebsiella spp. also decreased over time (1979–1990: 10.4%, 1991–1997: 6.3%, 1998–2010: 5.7%, P value 0.027). Enterococcus spp. was cultured more often in the most recent time period: 1998–2010 (1979–1990: 5.5%, 1991–1997: 4.6%, 1998–2010: 12.1%, P value 0.0008). No significant trends in prevalence over time were found for other bacterial species.
Several retrospective studies have evaluated the most common organisms isolated from both blood culture and necropsy specimens from foals with sepsis over the years [2, 3, 5, 11-20]. The subsets of foals included in some of these studies were also included in the current study [2, 17, 20]. The current study covers a longer time span and includes more bacterial isolates than previously published studies.
Although the relative frequency of isolation of different bacterial species varies between different studies, E. coli is the bacterial species isolated most commonly from foals with sepsis in all studies published to date. In the current study, the next most commonly isolated species, after E. coli, were Streptococcus spp., Actinobacillus spp., Klebsiella spp., Enterococcus spp., Staphylococcus spp. and Enterobacter spp. Potential reasons for differences in results between different studies include different time periods over which the studies were completed, geographical variation in bacterial populations, climate, differences in management practices on breeding farms that may select for or against infection with different bacterial species, differences in use patterns of antimicrobials for both prophylactic and therapeutic purposes, the specific age range of foals included in the studies, the specific samples for which culture information was included and differences in laboratory techniques used between different laboratories or within the same laboratory over different years.
Several aspects of the design of this study could have influenced the results obtained. Prior administration of antimicrobial drugs and hospitalisation before sampling are 2 factors that potentially influence prevalence of bacteria isolated from horses. Not all isolates included in the current study originated from samples collected at the time of admission; several originated from foals that had already been hospitalised for a variable period and others were isolated from samples collected at necropsy. The foals from which these isolates were cultured had typically, although not consistently, received antimicrobial treatment before the samples were obtained. Data on antimicrobial treatment before admission were not consistently available for all cases and could not therefore be taken into account in this study. Additionally, it is not known whether the site of sample collection has any influence on prevalence of bacterial isolates. Such an analysis was judged to be infeasible in this study because of the low number of isolates from most sites.
The relative frequency with which Gram-positive organisms were cultured from foals with sepsis increased significantly over time in this study. This finding is consistent with results of one previous study performed between 1986 and 2000 in Georgia, USA . The results of studies performed during the 1980s and the first half of the 1990s in the USA (Florida and Texas) and Australia, in which percentages of Gram-positive isolates of <20% were reported, and more recent studies from the USA (Florida and Pennsylvania) and Australia in which percentages of Gram-positive isolates of 25% or more were reported, also support a trend of increased prevalence of Gram-positive isolates in foals with sepsis [5, 11-16, 18]. The same trend has occurred in human medicine, where from 1979 through 1987, Gram-negative bacteria were the predominant organisms causing sepsis in man, whereas Gram-positive bacteria were reported to predominate in each subsequent year .
When comparing results of different prevalence studies it is important to recognise that the collection site of the specimen potentially influences the prevalence of Gram-positive bacteria. A study, published in 1989, showed that proportional distribution of species of bacteria isolated from blood samples was different from the distribution of bacteria isolated from internal organs at post mortem . The same study also showed that blood cultures were more accurate in identifying the presence of Gram-positive isolates than in detecting Gram-negative bacteria. Forty-three per cent of the isolated Gram-negative bacteria (including 60% of the E. coli bacteria) in that study went undetected by blood culture alone but were cultured at necropsy, as compared with only 10% of the Gram-positive bacteria that went undetected by blood culture alone.
Several studies that reported prevalence of bacterial isolates originating from foals with sepsis included only blood culture positive cases, which may have led to under-representation of Gram-negative bacteria in comparison with Gram-positive bacteria [14, 16, 18].
The temporal decrease in Gram-negative isolates in general and Enterobacteriaceae in particular, as a percentage of total isolates could potentially be the result of more extensive use in recent years of antimicrobial drugs such as aminoglycosides or third generation cephalosporins that have a predominantly Gram-negative spectrum of activity. This conclusion is supported by the finding in the current study that the most profound change occurred after prophylactic use of antimicrobials with a Gram-negative spectrum of activity became commonplace in foals in the USA in the late 1990s. A decrease in prevalence of Gram-negative enteric organisms in foals with sepsis was also reported in a study performed between 1982 and 2007 in Florida, USA . It has been hypothesised that the prophylactic use of antimicrobials in newborn foals helps prevent establishment of systemic infection with Gram-negative enteric bacteria that translocate across the intestinal barrier while it is still permeable for large molecules, such as immunoglobulin, but also bacteria .
Another possible explanation for the increase in prevalence of Gram-positive organisms is that the emergence and evolution of neonatal intensive care units and advanced critical care techniques in the last 2 decades has allowed critically ill foals that would have previously died or been subjected to euthanasia, to be treated. These foals have relatively long periods of hospitalisation and are therefore potentially more likely to acquire nosocomial infection, which in man often involves Gram-positive bacteria [22, 23]. Because most field-acquired infections are thought to occur through translocation of bacteria from the GI tract during the first few hours of life, they more often involve Enterobacteriaceae [4, 6, 10]. Unfortunately, historical data that would help distinguish between field-acquired and nosocomial infection was not available in the present study and therefore this hypothesis could not be tested. Further research will be necessary to support or refute this hypothesis.
Another potential explanation for the temporal increase in the proportion of Gram-positive isolates is the development of resistance to antimicrobial drugs among Gram-positive isolates . If this is the case, the recent emergence of Enterobacteriaceae that elaborate β-lactamases that inactivate extended spectrum β-lactam antimicrobials and render them resistant to third generation cephalosporins, raises the potential for Gram-negative organisms to again increase in prevalence as a cause of sepsis in the future [24, 25].
In the current study, Klebsiella spp. were isolated less frequently in recent years. Klebsiella spp. isolates were typically highly susceptible to amikacin and ceftiofur, drugs commonly used in equine practice. Other studies have reported a decrease in the proportion of Salmonella spp., Actinobacillus spp. and Streptococcus spp. isolates in recent years [18, 19]. These trends were not observed in the current study.
The most striking finding in the current study was the substantial increase in prevalence of Enterococcus spp. isolates between 1998 and 2010, a trend also reported in a study performed in Georgia, USA between 1996 and 2000 . The same trend has been seen in human medicine, where E. faecium has increased in importance as a cause of bloodstream infections . E. faecium is known to have a higher rate of antimicrobial resistance than other enterococci. In the current study, E. faecium was the most commonly isolated Enterococcus species (E. faecium n = 37, E. faecalis n = 21, other enterococci n = 21); therefore, it is possible that the increased prevalence of enterococci observed in this study could be the result of increased antimicrobial selection pressure for resistance. Enterococcus spp. are intrinsically resistant to several antimicrobial drugs and also readily accumulate mutations and exogenous genes that confer additional resistance through plasmids and transposons [27, 28]. Consequently, the susceptibility pattern of Enterococcus spp. isolates is unpredictable, which creates a therapeutic challenge for clinicians.
Extensive use of antimicrobials places strong selection pressure on bacterial isolates, favouring selection of resistant species such as Enterococcus spp. In a study performed in mice it was demonstrated that treatment with antibiotics perturbs the normal commensal microbiota and sets the stage for intestinal domination by bacteria associated with hospital acquired infections, including Enterococcus spp., preceding bloodstream infection . Enterococcus spp. are not only a threat to equine health; enterococci from animal origin can also act as donors of antimicrobial resistance genes for other pathogenic enterococci in man . Restrictive and well considered use of antimicrobial drugs is therefore crucial.
Because regional differences in prevalence of bacteria occur, the trends observed in the current study might be unique to the hospital where this study was performed. However, several trends are similar to those found in studies performed in other parts of the world, suggesting that the results of the current study may be applicable to other geographic locations [18, 19].
Collection of blood and other samples for bacterial culture remains an important component of the diagnostic work-up of foals suspected of having sepsis and also provides data to allow temporal trends in prevalence of individual bacterial species, as well as their antimicrobial susceptibility profiles, to be monitored. Observed trends form the basis for adjustment over time of first choice antimicrobial drugs to initiate treatment of foals with sepsis.
The increasing prevalence of Gram-positive bacteria as a cause of sepsis in neonatal foals, underlines the importance of including antimicrobial drugs active against both Gram-positive and Gram-negative bacteria in treatment protocols while awaiting the results of bacteriological culture and susceptibility tests. The increased prevalence of Enterococcus spp. should be of concern to clinicians because enterococci can have very unpredictable susceptibility patterns and can also act as donors of antimicrobial resistance genes to other bacteria. When managing neonatal foals, excellent hygiene and other measures are necessary to reduce the potential for nosocomial infection.
No competing interests have been declared.
Ethical review not required by this journal, it is a retrospective study based on clinical records.
This project was supported by the Center for Equine Health with funds provided by the State of California pari-mutuel fund and contributions by private donors.
The authors would like to thank Spencer Jang, Eileen Samitz and Dr Barbara Byrne from the UC Davis Microbiology Laboratory for their contributions to this study and Drs Astrid Watzin, Kirsten Wroolie and Maria DeCarlo for the work they have put into this study over the years. The authors would also like to thank the clinicians, residents, students and nursing technicians at the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California, Davis, USA, for their hard work and the excellent level of care they have provided to save hundreds of foals over the years.
All authors have made significant contributions to the completion of this study and have played an import role in drafting and/or revising the final article. All authors have approved the final version submitted for publication.
aBecton Dickinson and Co., Sparks, Maryland, USA.
bWampole, Cranbook, New Jersey, USA.
cCytel Software Corporation, Cambridge, Massachusetts, USA.