Microbiological Considerations in SepsisMicrobiological Considerations in SepsisCohen and Lynn

Jonathan Cohen1 and William A Lynn2

1Department of Infectious Diseases & Microbiology,Hammersmith Campus, Imperial College School of Medicine,2Department of Medicine, Ealing Hospital

Introduction

During the last ~ve to ten years the ~eld of sepsis hasbeen bedevilled by the problem of how to best de~nethe problem. “Septicaemia” (a term now no longerused) is an excellent example of a word everyoneunderstood but no-one could de~ne. In a series ofthoughtful articles [1–3], Bone and others pointed outthat the clinical syndrome classically associated withseptic shock could be mimicked by non-infective proc-esses such as acute pancreatitis. This gave rise to theconcept of SIRS (the systemic in_ammatory responsesyndrome) as an underlying pathophysiological expla-nation for what was seen at the bedside. However akey issue, which has since been hotly debated, is theextent to which infection is a sine qua non of the proc-ess. Vincent has argued cogently [4] that useful as itwas in our evolving concepts of sepsis, SIRS was even-tually misleading, in that it failed to de~ne suf~cientlyaccurately a reasonably homogeneous group of pa-tients. The view is now emerging (a view, incidentally,which we strongly endorse) that sepsis unequivocallyimplies the presence of infection. In everyday clinicalpractice it is not always possible to identify with cer-tainty what, or where, the infection is. Nevertheless,it follows that microbiological investigation is a crucialcomponent of the investigation and management of theseptic patient.

Epidemiology

A detailed discussion of the epidemiology of sepsis isbeyond the scope of this review, but there are a numberof general points which merit comment. First, approxi-mately 60% of cases of sepsis are associated with mi-crobiologically con~rmed infection, at least in the set-ting of a clinical trial. Second, almost all cases of sepsisare caused by bacterial infection. It is true that thereare some not dissimilar conditions caused by severeviral infections: dengue shock syndrome and some ofthe viral haemorrhagic fevers, for instance. But theseare special cases, and they are outside the usual spec-trum of cases seen in most intensive care units. Fungi—especially Candida species—can cause a sepsis syn-drome. In the EPIC study [5] more than 10% of blood-stream infections were caused by Candida spp., andeven Aspergillus infections can produce a similar pic-ture. Occasionally, a “typical” sepsis syndrome seems

to be caused by unexpected organisms: in one recentsepsis trial [6] cases associated with Legionella, Myco-bacteria and cytomegalovirus were documented. Itmay be arguable whether these were the cause of thedisease, but it is of interest that even in the morerigorous setting of a clinical trial, investigators wereidentifying these organisms in patients with carefullyde~ned sepsis syndrome. However, in the great ma-jority of cases sepsis is caused by conventional pyo-genic bacteria. Epidemiologic studies show that Gramnegative organisms account for 45%–65% of cases, andGram positive organisms cause the remainder. Thecommonest Gram negative isolates are the Entero-bacteriaceae (Escherichia, Citrobacter, Klebsiella,Enterobacter etc.), Pseudomonas species (usuallyPs.aeruginosa), and less often organisms such asStenotrophomonas maltophilia (previously Ps.malto-philia) and Acinetobacter. Amongst the Gram positivebacteria the usual culprits are Streptococcus pneumo-niae and Staphylococcus aureus; other isolates includeS.pyogenes and Staph.epidermidis (although there arealways concerns that coagulase negative staphylococcirepresent contamination rather than true infection).Interestingly, obligate anaerobic bacteria are regularlyreported, albeit in small numbers, as pure isolates fromblood cultures. They are rarely considered in the dif-ferential diagnosis, and this is of some signi~cance inthe choice of empirical antibiotic therapy. It is alsointeresting because their cell wall endotoxin differssustantially from that found in aerobic gram negativebacteria [7], yet the clinical features of sepsis seem nodifferent. Finally, it is helpful to consider the importantsites of infection. Accumulated data from clinical trialsshow that the blood, respiratory tract, surgical woundand abdominal cavity together account for most of theidenti~ed sites of infection [8]. Others, seen less often,are skin and soft tissue infections, the central nervoussystem, and the urinary tract (although it is well rec-ognised that even bacteraemic infections arising fromthe urinary tract uncommonly progress to establishedseptic shock).

Address for correspondence: Prof. J. Cohen, Department of Infec-tious Diseases & Microbiology, Imperial College School of Medi-cine, Hammersmith Hospital, Du Cane Rd., London W12 ONN,England.

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Sepsis 1998;2:101–106

© Kluwer Academic Publishers. Boston. Printed in U.S.A.

Why Evaluate Microbiology?

Part, but perhaps not all of the answer to this questionis self evident. It is helpful to consider a number ofdifferent aspects.

Diagnosis

An important role of microbiology is to establish thediagnosis. In the appropriate clinical setting, a pureisolate from a normally sterile site is usually suf~cientevidence to con~rm the diagnosis. Furthermore, posi-tive microbiology from a non-sterile site may providevaluable diagnostic information. Care is needed in bothcases: Staph.epidermidis in blood cultures, even inpure culture, does not necessarily provide an explana-tion for shock. Conversely, isolation of Pseudomonasaeruginosa from sputum or a tracheal aspirate mayindicate more than “colonising _ora”. Bacteriological~ndings often lag behind the clinical problem, butslowly this delay is being reduced. Modern blood cul-ture methodology, already widely available, will allowa “~rst look” after 6–8 hours of incubation, consider-ably earlier than the 18–24 hours we had been accus-tomed to.

Conventional bacteriological isolation is not the onlymeans of making a diagnosis. Many immunologicaltests are in routine use and widely available, and areperhaps not used as often as they might be. A pneumo-coccal antigen test, for example, can be performed onblood, urine and empyema _uid. It may prove positiveat different times (the urine often remains positivelong after the blood is negative, for instance) and maybe positive when cultures are negative because of priorantibiotic use. Non-culture techniques based on thepolymerase chain reaction are still mainly experimen-tal, but these too are beginning to appear in the diag-nostic laboratory. It is unlikely that they will be able toprocessed in less than several hours.

Prognosis

Certain bacteria have traditionally been associatedwith a bad prognosis. Pseudomonas aeruginosa, forinstance, is often thought of as a particularly ominousisolate, and more recently Streptococcus pyogenes hasbeen associated with especially severe complications ofthe so-called “streptococcal toxic shock syndrome” [9].Two large recent studies have provided some morespeci~c details.

Weinstein et al. [10] studied all adult inpatients withpositive blood cultures over a 12-month period. Al-though coagulase negative staphylococci were thecommonest isolates, only 12% were thought to be clini-cally signi~cant. The most common pathogens wereS.aureus, E.coli, K.pneumoniae and Enterococcus spe-cies. Certain micro-organisms were associated with asigni~cantly greater mortality. Fungaemia (mostlyCandida infections) had a mortality of 36% and a rela-

tive risk (RR) of death of 6.54 (p ,0.001). Amongst theGram negative bacteria, Enterobacteria other thanE.coli had a mortality of 25% (RR 4.53 p ,0.01) al-though interestingly, Ps.aeruginosa was associatedwith a RR of 3.04 (p 5 NS). Of the Gram positivebacteria, S.pneumoniae bacteraemia had a RR of 3.22,S.aureus of 2.18. In contrast, bacteraemia due to co-agulase negative staphylococci had a mortality of just5.5% (RR 1.0, p 5 0.05). In a multivariate analysis,infection with fungi and Enterobacteriaceae other thanE.coli was associated with a RR of 2.27 (p , 0.01).

In this study, blood pressure at the time the ~rstpositive blood culture was obtained was used as a sur-rogate for shock. Approximately 14% of patients werehypotensive at this point, and the distribution of or-ganisms amongst the hypotensive and normotensivegroups did not differ. Not surprisingly, mortality washigher in the hypotensive group. One consistent factorwhich emerged was the poor prognosis associated withpolymicrobial sepsis; not only were these cases statis-tically more likely to present with hypotension (p ,0.05, RR 1.58) but the mortality was twice as high(34.2% vs 15.7%, p , 0.001 RR 2.16).

In the second study [11], the approach differed, theintention was to evaluate patients who acquired a noso-comial blood stream infection, and the methodologyexcluded episodes thought to represent contamination.The microbiological ~ndings were similar to those ofthe ~rst study: coagulase negative staphylococci (caus-ing line infections), S.aureus, E.coli, Klebsiella spp andother Gram negative rods, and Candida spp., were thecommonest isolates. Pathogen-speci~c 28 day mortal-ity rates ranged from high values for Candida spp. of35% (OR 1.98 95% CL 1.36–2.28, p 5 0.0002) andPs.aeruginosa (31%, OR 1.58 95% Cl 1.02–2.43 p 50.024) to 17% for coagulase negative staphylococci.Multiple logistic regression procedures were used tomodel factors that independently in_uenced 28 daysurvival. The microbiological factors identi~ed werepneumonia as a source of infection (OR 2.74; 95% CI1.87–4.00; p , 0.0001), infections due to Candida spp.(OR 1.84; 95% CI 1.22–2.76; p 5 0.035), and polymicro-bial infections (OR 1.68; 95% CI 1.22–2.32; p 5 0.0014).Although not statistically signi~cant in the model, in-fections due to Ps.aeruginosa were associated with apoor outcome (OR for 28 day mortality 1.04; 95% CI0.96–1.13; p50.31).

In summary, these two reports, which are broadlyrepresentative of a large body of data, con~rm thatmicrobiological variables are independently and signi-~cantly associated with the outcome of sepsis. Thisobservation begs the question of mechanism, perhapsa re_ection both of innate virulence determinants incertain bacteria and relative defects in defence mecha-nisms in some hosts. Nevertheless, it is clear thatmicrobiological ~ndings do provide prognostic infor-mation, and therefore have a bearing on clinical trialdesign (see below).

102 Cohen and Lynn

Treatment

Nearly 20 years ago Kreger and others [12] showed thatappropriate antibiotic therapy reduced the fatality rateof patients with bacteraemia by approximately 50%, ir-respective of the severity of their underlying disease.Early appropriate antibiotic therapy had a similarlysalutary effect on the development of shock. More re-cently, a study [10] evaluated the appropriateness of an-tibiotic therapy at three points: ~rst, given empirically,second, after the result of the bacterial culture wasavailable, and third, after the susceptibility tests wereknown. Although the numbers were small, there was aclear survival disadvantage from inappropriate ther-apy, especially in the early stages of empirical treat-ment. It is particularly interesting to compare the out-come of patients in whom treatment was changed as aresult of the susceptibility data. The mortality of the 620patients who had appropriate therapy at all three timepoints was 10.5% (RR 1.0); in comparison, the mortalityof the 35 patients who had inappropriate therapy aftersusceptibility data were available was 20% (RR 1.91).Thus, an obvious and tangible bene~t of microbiologicalresults is the ability to prescribe the correct treatment.A secondary bene~t is the opportunity to avoid the un-necessary use of drugs such as vancomycin or imi-penem, and so to lessen the burden of antibiotic resis-tance, a topic which is discussed in more detail below.

Although it is not surprising that the correct choiceof antibiotics improves the outcome, it is much lessclear whether the presence, or type of infection wouldhave any in_uence on the success of non-antimicrobialforms of treatment. It may well turn out that infectionis an important variable predicting the success ofmediator-directed therapies. A recent analysis of alarge phase II clinical trial of a type I TNF-receptormolecule revealed that there was a statistically signi~-cant treatment effect in patients with microbiologicallycon~rmed infections, an effect not seen in those whoseinfections were diagnosed on clinical ground (J Cohenet al., manuscript submitted). Some forms of adjuvanttherapy have been targeted at speci~c organisms. Anti-endotoxin strategies, of which there are several nowunder active investigation, are directed at patientswith Gram negative infections. The initial clinical trialsof bactericidal/permeability-increasing protein (BPI)for instance, have been done in children with meningo-coccal disease [13]. Next, some agents which have beenevaluated in a mixed group of septic patients with bothGram negative and Gram positive infections may turnout to be more effective in one than the other. For in-stance, a study of a PAF antagonist [14] seemed moreactive in Gram negative than in Gram positive infec-tions. Conversely, a recent trial of a type II TNF recep-tor molecule [15] turned out to have a deleterious effect,especially in patients with Gram positive infections. Arecent review of the evidence (S. Opal & J. Cohen, CritCare Med, in press) concluded that there was a growingbody of data suggesting that there were important dif-

ferences between Gram negative and Gram positiveinfections, and that these might have some bearing onthe ef~cacy of anti-mediator strategies.

Epidemiology and infection control

High quality microbiological data provide an essentialinfrastructure to allow logical antibiotic prescrib-ing. Antibiotic resistance patterns differ substantiallyaround the world, and even between different units inthe same city. Furthermore, these patterns are subjectto constant change. Since the majority of antimicrobialprescribing is done empirically, before susceptibilitydata are available, it is clear that knowledge of the localpatterns of resistance is essential. As noted above, theuse of “appropriate” antibiotic therapy is an importantvariable in determining outcome from sepsis, thus un-derlining the signi~cance of this information in terms ofcomparability of treatment outcomes in a clinical trial.

For the same reasons, microbiological data are avital part of the infection control process, the “sentinelchickens,” as it were, providing warning of possiblebreakdowns of sterility or hygiene practices.

What then should be the approach to routine micro-biological surveillance? It is generally accepted thatroutine, hospital-wide surveillance is impractical, butequally, that directed surveillance in special units isworthwhile. Certainly it is valuable to maintain a data-base, continually updated, of sensitivity data from im-portant isolates such as blood cultures. Screening issometimes carried out for epidemiological reasons, forinstance to look for carriage of methicillin-resistantStaph.aureus or vancomycin-resistant enterococci.This is often done when there is an outbreak, butshould it be routine? Some units screen all transfersfrom outside the hospital, or from outside a de~nedgeographic area. Whether all patients entering a unitshould be routinely and repeatedly swabbed in order todocument their “normal” _ora, irrespective of whetherthey are clinically infected, is more debatable, and doesnot seems to have been formally studied. Several stud-ies have provided good evidence that the intensity ofcolonisation with Candida is closely related to the sub-sequent development of Candida sepsis [16,17], and itwould certainly seem reasonable to screen patients atrisk of developing systemic candidiasis. The merit ofroutine bacteriological screening is less clear. However,there are data which suggest that up to three quartersof all ICU infections are caused by organisms whichhad previously colonised the patient, and based on this,some units do routinely swab endotracheal tubes andtake urine samples, for instance, two or three times aweek [18]. However, it should be emphasised that for-mal cost-effectiveness studies have not been done.

Clinical trials

Infection is an integral component of sepsis. It followstherefore that the correct identi~cation of infection

Microbiological Considerations in Sepsis 103

should be a sine qua non of the methodology of clinicaltrials in sepsis. Interestingly, this has seldom been thecase. This derives not so much from an unwillingnessto recognise the importance of appropriate microbio-logical methods, but rather because there has beenlittle attention given to them.

Appropriate microbiological procedures are impor-tant for two broad reasons: ~rst, they will ensure accu-racy and comparability of the data, and second, theymay be important in interpreting the results (Table 1).

Like all laboratory investigations, microbiologicaltests will provide different results depending on howthey are done. To non-microbiologists, a blood culturemay seem a rather unambiguous procedure: a sample ofblood is obtained and if the patient is bacteraemic anorganism will be isolated. In fact, there are many vari-ables to consider including the volume of blood ob-tained, the nature of the culture medium, the durationof incubation and so on. These variables can make thedifference between whether a culture is eventually re-ported as positive or negative. It is obvious then thatthese methodological considerations (which apply to allspecimens, not just blood cultures), must be stand-ardised between centres to ensure a reasonably homo-geneous population in the study. For example, if a studyis to be carried out in patients with Gram negativepneumonia or meningococcal sepsis we need to knowthe criteria on which the diagnosis was made. The ques-tions become especially important when the data areanalysed. Several of the large clinical trials have notedthat patients in microbiologically-de~ned subsets haveresponded favourably. A good example was the largeHA-1A study [19]; the analysis suggested that it wasonly active in patients with Gram negative bacterae-mia. More recently, the trial of the p75 soluble TNFreceptor reported that an adverse outcome seemed tobe associated especially with Gram positive infections[15], while in contrast, analysis of a Phase II study withthe type I p55 TNF receptor fusion protein suggests

that it was more effective in microbiologically de~nedinfections (J. Cohen et al., manuscript submitted).

Finally, it is clear that accurate microbiology is es-sential to the process of evaluating appropriateness oftherapy, a crucial variable in determining outcome insepsis trials.

Given the evident importance of the factors summa-rised in Table 1, it is interesting to re_ect on just howwell microbiological methodology has been consideredin the protocols of the trials that have been done. Infact, there is considerable variation over such funda-mental issues of how and when to take blood cultures,how to carry out the microbiological procedures, howinfection should be de~ned and so on. Not only is therelittle consensus, but it is clear that in some, otherwisecarefully designed trials little thought has been givento the microbiological methods.

In the following section we will take as an exampleblood cultures, since they are the single most impor-tant investigation in the context of sepsis, and discusssome of these methodological issues in more detail.

Microbiological Methods inBacteraemia

Many factors have been shown the in_uence the ef~-cacy, and/or the speed of recovery of bacteria from bloodcultures (Table 2). Some of these are of a technical na-ture and will be of principal interest to microbiologists.Furthermore, it will often not be possible to standardisethe methods used exactly between different centres,since each laboratory will have its own system and willnot be able to change that just for the purpose of aclinical trial. However, there are some variables thatcan be easily regulated and which have been shown tohave a signi~cant impact on bacteraemia rates.

Contamination rates

When blood cultures are reported to be positive from aseptic patient, there is a strong inclination to assumethat they are the cause of the patients’ illness, even ifthe organisms are of low virulence, such as coagulasenegative staphylococci. In fact it is often impossible tobe certain if the positive cultures represent contamina-tion or true infection; nevertheless it is clear that cer-tain simple practices have been shown to reduce the

Table 1. The importance of microbiological procedures in thedesign and interpretation of clinical trials in sepsis

A. ACCURACY AND COMPARABILITY OF THE DATA1. The use of standardised microbiological methods to ensure

accurate diagnoses and comparability between centres.2. To ensure comparability in entry criteria, and/or to de~ne

microbiological criteria for study entry.3. To take account of differences between units in the

pattern of infection and/or antibiotic susceptibility data.

B. INTERPRETATION OF THE RESULTS4. To be able to con~rm the presence and nature of infection.5. To be able to make an assessment of the appropriateness

of antibiotic therapy.6. To establish if the presence, of the type of infection has

any in_uence on the response to the investigational agent.7. To ensure the accurate detection of complications, such as

superinfections.

Table 2. Factors which may in_uence the ef~cacy, or speed ofrecovery of bacteria from blood cultures

Site of venepunctureTechnique of venepunctureVolume of blood culturedCulture medium, and additivesCulture conditionsDuration of cultureDetection system

104 Cohen and Lynn

likelihood of contamination, usually from skin organ-isms. For instance, protocols should specify that wher-ever possible, blood cultures should not be drawn fromthe femoral site, through areas of abnormal skin orfrom indwelling catheters. Careful skin preparation,disinfection of the tops of blood culture bottles andimmediate inoculation of the blood culture set shouldresult in a contamination rate of less than 3% [20].

Optimising detection - blood volume.

The volume of blood cultured has a signi~cant impact onthe recovery of bacteria from the culture. In most bac-teraemias there are not many organisms in the blood; atypical ~gure is 10 colony forming units per millilitre(cfu/ml), although it may be as low as 0.1 cfu/ml [20,21].Hence, inadequate sampling may fail to detect clinicallysigni~cant bacteraemia. Mermel and Maki [22] showedthat there was a signi~cant difference in the detection ofbacteraemia when volumes of 8.7 ml were comparedwith 2.7 ml (92% vs 69%, p , 0.001). This and otherstudies have con~rmed that the positive detection rateincreases by about 3% for every additional millilitre ofblood cultured.

In a recent study, Weinstein et al. compared theef~cacy of 5 ml versus 10 ml volumes of blood cultured inone of the standard, modern, semi-automated blood cul-ture machines, the BacT/Alert system [23]. The studyexamined over 13,000 pairs of blood cultures from whicha total of almost 800 organisms were isolated. The over-all recovery of organisms from the 10 ml set exceededthat from the 5 ml set by 7.2% (p , 0.001). This differ-ence was most marked for E.coli (p , 0.01) and otherEnterobacteriaceae (p , 0.001). Furthermore, the bac-teraemias were detected signi~cantly earlier in the 10ml set than in the 5 ml set. Thus, quite apart from theclinical utility of having a faster result, the simple vari-able of the volume of blood drawn can have a signi~cantimpact on whether a blood culture is reported positiveor not: the implication of this variable for the interpreta-tion of clinical trials of sepsis is obvious.

The total volume of blood that should be obtainedfrom adults is 20–30 ml; there is no bene~t in exceedingthis. Since most commercial systems will allow up to amaximum of about 15 ml, a practical recommendationis that protocols should specify that at least two sepa-rate blood cultures, each of 10 ml., should be obtained.Maximum yield will be obtained with three cultures,which will detect 99% of bacteraemias [24]. There is noneed to do more than this, and there is no advantage ofarterial cultures over venous cultures. There are nodata on the optimum period of time over which thecultures should be obtained, but in practice, there areno grounds to delay, and antibiotics should be givenpromptly after the blood cultures have been obtained.

Laboratory methodology

This has been reviewed elsewhere [25] and is beyondthe scope of this paper. It is worth noting though that a

variety of modi~cations of the ways in which the labora-tory processes the samples can have an impact on thespeed with which results are available, and/or the prob-ability of detecting certain organisms, such as Candidaspp., The in_uence of this variability on clinical manage-ment (and hence, the speed with which appropriate an-tibiotics are given, for example) has not really been con-sidered. Presumably any effect would not be enough tohave a major impact on a clinical trial, although investi-gators should be aware of these differences as a possibleexplanation for variable data between different centres,especially in international trials.

Interpreting the result

When is a positive blood culture signi~cant? The an-swer, presumably, is when it correlates with a clinicalsyndrome of sepsis but this is a circular argument.From a microbiological standpoint it would be usual toregard a high-grade pathogen (E. coli, Staph.aureus,etc.) as signi~cant even if it were isolated from a singlebottle of one set of cultures. Conversely, low-gradeorganisms such as coagulase negative staphylococci ordiphtheroids would normally have to be obtained fromat least two sets. It is interesting to re_ect that evensuch a fundamental issue as this has often not beende~ned in the clinical trial protocols in sepsis.

Conclusions

Infection is an integral component of sepsis, and itfollows that the diagnosis of infection is a key compo-nent in the investigation and management of the septicpatient. Microbiological issues in the design of clinicaltrials have probably received inadequate attention inthe past, and variability in microbiological methods canhave a signi~cant impact on various aspects of trialoutcome. It will be important that protocols are devel-oped and consensus is reached in the area of microbio-logical investigation and antimicrobial treatment, justas it is in areas such as haemodynamic monitoring orventilator management.

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