Evaluation of Techniques for Detection of Carbapenem-Resistant Klebsiella pneumoniae in Stool Surveillance Cultures

Screening for gastrointestinal colonization with multidrug-resistantnosocomial pathogens is an important component of infectioncontrol protocols. In the New York City region, carbapenem-resistantKlebsiella pneumoniae strains, which harbor the KPC carbapenem-hydrolyzingß-lactamase, have rapidly emerged. The potential utilityof screening medium, which involved using 10-µg imipenemdisks, was investigated. The method of placing a sample froma fecal surveillance culture into broth containing an imipenemdisk appeared to have the greatest sensitivity for detectingKPC-producing K. pneumoniae. Gastrointestinal colonization withtwo other carbapenem-resistant nosocomial pathogens, Pseudomonasaeruginosa and Acinetobacter baumannii, was also detected usingthis method. Placing fecal surveillance specimens into brothcontaining an imipenem disk is an easy method for screeningsamples for carbapenem-resistant nosocomial pathogens.

INTRODUCTION

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Introduction

Controlling the spread of antibiotic-resistant nosocomial pathogensinvolves a combination of antibiotic control measures and effectiveinfection control strategies. For example, recommendations forthe control of vancomycin-resistant enterococci include reductionof vancomycin use, strict adherence to infection control practices,and early detection by screening for gastrointestinal colonization(5). Implementation of these measures has successfully controlledthe spread of this pathogen (14). The availability of commerciallyprepared media that can be used to screen rectal cultures forvancomycin-resistant enterococci facilitates laboratory detection.Similarly, effective control strategies for controlling membersof the family Enterobacteriaceae with extended-spectrum ß-lactamases(ESBLs) have involved aggressive infection control protocolsthat included screening for gastrointestinal colonization (11).Unfortunately, suitable media for screening fecal cultures forresistant gram-negative pathogens are not commercially available.Studies examining rates of gastrointestinal carriage of cephalosporin-resistantEnterobacteriaceae strains have used locally prepared media,including agar supplemented with cefotaxime (7, 10, 11, 13).

The recent and rapid spread of carbapenem-resistant Klebsiellapneumoniae, due to the presence of the carbapenem-hydrolyzingß-lactamase KPC, in the New York City region has beenalarming (1, 2, 4, 16). This pathogen presents a formidablechallenge because of its high degree of resistance to virtuallyall classes of antibiotics (4), and controlling their spreadis of utmost importance. In this report, we investigate potentiallaboratory procedures that could be used by clinical microbiologylaboratories for the screening of fecal specimens for theseresistant bacteria.

MATERIALS AND METHODS

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Materials and Methods

Preliminary studies. Six previously characterized clinical strains of carbapenem-resistantK. pneumoniae, known to possess bla_KPC-2, were included in experimentsto assess the sensitivity of three potential screening methods(1, 4). These six isolates represented the two major ribotypes(two isolates each) involved in outbreaks in New York City,and two isolates from unique ribotypes. Susceptibility testingusing Etest, the disk diffusion technique, and the broth microdilutionmethod was performed according to established recommendations(6).

Starting with an initial inoculum of $~$ 5 x 10⁵ CFU/ml (range,1 x 10⁵ to 1.7 x 10⁶ CFU/ml), serial 10-fold dilutions of thesix isolates were made in normal saline. To include the effectof possible interference from other carbapenem-resistant organismsthat can inhabit the gastrointestinal tract, the following organismswere added to each dilution ( $~$ 1 x 10⁴ CFU/ml): a clinical isolateof imipenem-resistant Pseudomonas aeruginosa, a clinical isolateof vancomycin-resistant Enterococcus faecium, and Candida albicansATCC 90028. The culture mixture of each dilution was then processedin three ways. For method 1, 100 µl of the culture mixturewas placed in 5 ml of tryptic soy broth containing a 10-µgdisk of imipenem. Following an overnight incubation at 37°C,100 µl was streaked onto MacConkey agar and incubatedovernight; the presence or absence of lactose-fermenting gram-negativerods was then recorded. For method 2, 100 µl of the culturemixture was placed in 5 ml of MacConkey broth containing a 10-µgdisk of imipenem. Following an overnight incubation at 37°C,100 µl was streaked onto MacConkey agar and incubatedovernight; the presence or absence of lactose-fermenting gram-negativerods was then recorded. For method 3, 100 µl of the culturemixture was placed in 5 ml of tryptic soy broth. Following anovernight incubation at 37°C, 100 µl was streakedonto MacConkey agar and an imipenem disk was applied. Isolatesof K. pneumoniae growing within the zone of inhibition (withina zone diameter of $≤$ 15 mm) were recorded. Prior studies demonstratedcomparable zones of inhibition for imipenem against P. aeruginosaATCC 28753 when Mueller-Hinton and MacConkey agars were used(data not shown).

Stool surveillance studies. Surveillance rectal cultures are routinely obtained from allpatients in a 10-bed medical-surgical intensive care area aspart of an infection control program to identify vancomycin-resistantenterococci. The cultures are performed upon admission to theintensive care area and once weekly during the patients' stay.Rectal swabs are placed in BBL CultureSwab Plus culturettes(Becton Dickinson, Sparks, MD) containing 5 ml of Amies geltransport medium. During the month of February 2005, an aliquotof transport medium was collected from each culturette beforeit was discarded. Imipenem-resistant K. pneumoniae strains wereendemic in the intensive care area during that period. To ensurean equal inoculum for each method, 100 µl of the transportmedium was placed into 5 ml of tryptic soy broth and processedaccording to methods 1 and 3 described above. All gram-negativepathogens in method 1, and all growing within a zone diameterof $≤$ 15 mm in method 3, underwent imipenem susceptibility testingusing the Etest methodology. All imipenem-resistant isolateswere then identified using the API 20E system (bioMérieux,France) and screened by PCR for the presence of bla_KPC usingpreviously defined primers and conditions (1, 4). Amplifiedbla_KPC products were identified with bidirectional sequencing,as previously described (1, 4).

This study was approved by the Institutional Review Board atSUNY-Downstate Medical Center.

RESULTS

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Results

Preliminary studies. All six clinical isolates used in these studies were intermediateor resistant to imipenem using the recommended inoculum forbroth micodilution susceptibility testing (Table 1). However,three isolates had MICs of imipenem that were highly dependenton the inoculum used, a phenomenon frequently observed in KPC-possessingK. pneumoniae (4). The lowest limits of detection of six clinicalisolates of K. pneumoniae with bla_KPC-2 by the three methodsare reported in Table 1. Isolates that had MICs of imipenemthat were highly inoculum dependent were more difficult to detect,regardless of the method, whereas isolates that had high MICsof imipenem regardless of inoculum were more easily detected.Methods 1 and 2 yielded comparable results. Method 3 was inferiorin detecting three of the isolates (requiring at least 1 log₁₀CFU/ml more than the other two methods), superior in the detectionof one isolate, and equivalent for the remaining two isolates.Because methods 1 and 2 yielded comparable results, evaluationof the clinical samples was performed by only methods 1 and3.

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TABLE 1. Comparison of three methods for the detection of six clinical isolates of carbapenem-resistant K. pneumoniae

Stool surveillance studies. Fifty-one stool surveillance cultures from 33 patients underwenttesting by methods 1 and 3. For method 1, 13 lactose-fermentinggram-negative bacilli were recovered from 12 samples. Of these13 isolates, five were imipenem susceptible (MIC range, 0.25to 3 µg/ml) and eight were resistant to imipenem (MIC,>32 µg/ml). All eight resistant isolates were identifiedas K. pneumoniae with bla_KPC-2. In addition, 29 non-lactose-fermentinggram-negative bacilli were recovered. Ten were found to be imipenemsusceptible (MIC range, 0.25 to 4 µg/ml). Of the remaining19 isolates, 17 were P. aeruginosa and two were Acinetobacterbaumannii, all with imipenem MICs of >32 µg/ml.

When the clinical samples were evaluated using method 3, fourcarbapenem-resistant K. pneumoniae strains, all with bla_KPC-2,were identified. In addition, 19 imipenem-resistant isolatesof P. aeruginosa were identified. Since only isolates growingclose to the imipenem disk were evaluated, none of the isolatesevaluated were found to be imipenem susceptible. A. baumanniiwas not recovered from any of the samples using method 3.

In total, eight samples had imipenem-resistant K. pneumoniae(four by both methods and four by method 1 only). In addition,23 samples had imipenem-resistant P. aeruginosa. Of these, 13were positive by both methods, four were positive by method1 only, and six were positive by method 3 only.

Fourteen of the 33 patients had more than one surveillance sampletaken during the study period. The cultures from these patientswere either all positive or all negative in 7 of 14 cases. Forthe remaining seven patients, the initial culture was negativewhereas the subsequent culture(s) grew carbapenem-resistantK. pneumoniae or P. aeruginosa. The latter patients may havebecome colonized after the initial culture was taken. No patientswith an initially positive culture had a subsequent culturethat was negative. Thirteen of the 33 patients had culturesthat were all negative. All 13 patients had other (nonsurveillance)cultures taken by the medical team as part of their routinecare. None of these cultures grew carbapenem-resistant K. pneumoniaeor P. aeruginosa. However, 3 of the 13 patients had routinecultures that grew carbapenem-resistant A. baumannii (two fromrespiratory cultures and one from blood culture).

DISCUSSION

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Discussion

Detection of antibiotic-resistant nosocomial pathogens (e.g.,vancomycin-resistant enterococci or Enterobacteriaceae strainswith ESBLs) that commonly colonize the gastrointestinal tractis an integral component of successful infection control protocols(5, 11, 14). Commercially prepared media are available thatcan be used by clinical laboratories for the detection of vancomycin-resistantenterococci. However, because of the instability of many ß-lactamsin culture media, no such media exist for the detection of resistantgram-negative pathogens. The difficulty of screening fecal surveillancecultures for bacteria with ESBLs has undoubtedly been an impedimentin our efforts to control the spread of these pathogensin the New York City region (15).

Since 2001, our region has also witnessed the rapid spread ofcarbapenem-resistant K. pneumoniae; carbapenem resistance inthese isolates is due to the presence of KPC, an efficient classA carbapenem-hydrolyzing ß-lactamase (2, 4, 16). Becausethese pathogens are resistant to virtually all commonly usedantibiotics, including ß-lactams, fluoroquinolones,aminoglycosides, and not infrequently polymyxins (4), controllingtheir spread is of utmost importance. As with vancomycin-resistantenterococci and ESBL-possessing Enterobacteriaceae, relianceon cultures taken from patients with clinically suspected infectionmay fail to identify patients harboring KPC-possessing K. pneumoniae(2). In outbreak settings, screening for asymptomatic colonizationof the gastrointestinal tract will be necessary to identifypatients with these pathogens, so that proper infection controlefforts can be instituted.

In this report, the medium that consisted of 5 ml of broth containinga 10-µg imipenem disk resulted in the highest recoveryof KPC-possessing K. pneumoniae from stool surveillance cultures.This method can be easily performed by clinical laboratories.It was apparent that the targeted concentration of imipenem(2 µg/ml, assuming 100% diffusion of the antibiotic) wasnot achieved with this method, as several of the pathogens isolatedby this method had imipenem MICs of between 0.25 and 4 µg/ml.Confirming imipenem resistance by a disk diffusion assay priorto identification of these isolates would minimize unnecessarywork, although it would delay reporting by another 24 h. Increasingthe number of imipenem disks placed into the broth may reducethe likelihood of recovering imipenem-susceptible bacteria.The alternative method (plating an overnight growth of the surveillanceculture onto MacConkey agar with an imipenem disk and identifyingonly the isolates near the disk) eliminated the problem of therecovery of carbapenem-susceptible bacteria but appeared lesssensitive for the detection of KPC-possessing K. pneumoniae.bla_KPC has been found in other bacteria, including Salmonellaenterica, Klebsiella oxytoca, and Enterobacter spp. (3, 8, 12,17). An assessment of these methods for detecting other KPC-producingEnterobacteriaceae in stool surveillance cultures will haveto be performed.

Our region has also been beset with the clonal spread of twoother carbapenem-resistant pathogens, A. baumannii and P. aeruginosa(9). An unexpected finding in our study was the relatively largenumber of stool samples harboring these isolates, since theyare not commonly recognized as inhabitants of the gastrointestinaltract. It appears that the laboratory methods outlined in thisstudy will also be useful in detecting gastrointestinal colonizationwith these pathogens and assisting infection control efforts.

FOOTNOTES

* Corresponding author. Mailing address: Division of Infectious Diseases, Box 77, SUNY Downstate, 450 Clarkson Ave., Brooklyn, NY 11203. Phone: (718) 270-3790. Fax: (718) 270-2468. E-mail: dlandman{at}downstate.edu.

REFERENCES

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