Nasopharyngeal Carriage of Klebsiella pneumoniae and Other Gram-Negative Bacilli in Pneumonia-Prone Age Groups in Semarang,Indonesia

Helmia Farida,a Juliëtte A. Severin,b M. Hussein Gasem,c Monique Keuter,d Peterhans van den Broek,e Peter W. M. Hermans,f,g

Hendro Wahyono,a Henri A. Verbrughb

Department of Microbiology, Faculty of Medicine, Diponegoro University, and Dr. Kariadi Hospital, Semarang, Indonesiaa; Department of Medical Microbiology andInfectious Diseases, Erasmus University Medical Centre, Rotterdam, the Netherlandsb; Department of Internal Medicine, Dr. Kariadi Hospital, and Faculty of Medicine,Diponegoro University, Semarang, Indonesiac; Department of General Internal Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlandsd;Department of Infectious Diseases, Leiden University Medical Centre, Leiden, the Netherlandse; Nijmegen Institute for Infection, Inflammation, and Immunity (N4i),Nijmegen, the Netherlandsf; Laboratory of Paediatric Infectious Diseases, Radboud University, and Nijmegen Medical Centre, Nijmegen, the Netherlandsg

Gram-negative bacilli (GNB) cause many cases of pneumonia in Indonesia. We investigated nasopharyngeal carriage of GNB inSemarang, Indonesia. Klebsiella pneumoniae carriage in adults (15%) was higher than in children (7%) (P � 0.004), while that ofother GNB was comparable. Poor food and water hygiene are determinants of carriage of these bacteria.

Worldwide, Streptococcus pneumoniae is the most commoncause of community-acquired pneumonia (CAP). How-

ever, in low-to-middle-income countries, Gram-negative bacilli(GNB) are reported as frequent causes of CAP (1). In a recentstudy on CAP in Indonesia, Klebsiella pneumoniae was the mostcommon etiological agent (our unpublished observation). Sincenasopharyngeal colonization may precede pneumonia, as for S.pneumoniae (2), we investigated nasopharyngeal carriage of K.pneumoniae and other GNB among children and adults comparedto that of S. pneumoniae in Indonesia. Subjects were healthy chil-dren (6 to 60 months old) and adults (45 to 70 years old) living inthe city of Semarang on the island of Java in Indonesia, withoutrespiratory symptoms and without antibiotic consumptionwithin the last 3 days. Subjects were recruited from all 16 districtsof Semarang by cluster random sampling from February to April2010.

Nasopharyngeal swabs were obtained and transported inAmies (COPAN, Italy) and then were inoculated on 5% sheepblood agar with gentamicin (5 mg/liter) and on MacConkey agar.The plates were incubated at 35°C in 5% CO2. Identification andantimicrobial susceptibility tests for GNB were performed usingVitek 2 (bioMérieux, Marcy l’Etoile, France). Identification of S.pneumoniae was performed using the optochin test (Oxoid, Bas-ingstoke, United Kingdom). Control strains were included, andCLSI guidelines were applied.

A questionnaire was developed to identify determinants of car-riage of these bacteria. Data on demography, water supply (fordrinking, preparing food, and bathing), food hygiene and housesanitation (crowding, flies, and exposure to smoke from cigarettesand mosquito coils), were collected. Water hygiene was consid-ered poor when water other than tap water or bottled water wasused. Food hygiene was considered poor if food was bought fromstreet vendors. Crowding was considered if the ratio of bedroomspace per family member was �4 m2 (3). Univariate analysis wasdone with chi-square or Fisher’s exact test when appropriate, fol-lowed by logistic regression using SPSS 17 (SPSS Inc., Chicago, IL,USA). Confidence interval (CI) was calculated at the 95% level(CI95), and P values of �0.05 were considered significant. Writteninformed consent was obtained from the subjects or their caregiv-

ers. The study was approved by the Ethical Committee of the Fac-ulty of Medicine, Diponegoro University.

A total of 253 adults and 243 children participated in the study;approximately 25% resided in the suburbs, and the majority wereliving under hygienically restricted circumstances regarding theirfood and water supplies. Crowding and exposure to smoke werecommon (Table 1).

The carriage rate of K. pneumoniae and other GNB was 11%(CI95, 7 to 14%) and 19% (CI95, 12 to 23%), respectively. K. pneu-moniae carriage in adults (15%) was higher than in children (7%)(P � 0.004), while that of other GNB was comparable (Table 2).Carriage of S. pneumoniae was 43% in children and 11% in adults(P � 0.000). Combined carriage of these bacteria was common.The presence or absence of one species was not significantly cor-related to that of the other species (P � 0.1). The prevalence ofcarriage of these bacteria varied significantly by city districts andtended to be higher in the suburban and eastern parts of the city.

Fifty-four K. pneumoniae strains were isolated from 54 sub-jects. One isolate was resistant to cefotaxime and ceftriaxone, butthe presence of an extended-spectrum beta-lactamase could notbe confirmed. The remaining strains were susceptible to commonantibiotics, including cefuroxime, but excluding nitrofurantoin(43% resistant).

One-hundred seven GNB strains other than K. pneumoniaewere found in 96 (19%) subjects (49 children and 47 adults).The three most frequently isolated genera were Pseudomonas,Enterobacter, and Acinetobacter (Table 3). All members of thefamily Enterobacteriaceae other than K. pneumoniae weresusceptible to carbapenem, and 95% of the strains were sus-ceptible to piperacillin-tazobactam, expanded-spectrum ceph-alosporins, gentamicin, meropenem, and ciprofloxacin. All

Received 5 March 2013 Accepted 6 March 2013

Published ahead of print 13 March 2013

Address correspondence to Helmia Farida, helmia_farida@yahoo.com.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.00589-13

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Pseudomonas isolates were susceptible to tobramycin, mero-penem, and ciprofloxacin, 67% were susceptible to ceftazi-dime, and 87% were susceptible to colistin.

Multivariate analysis showed that being an adult (odds ratio[OR], 2.2; CI95, 1.1 to 4.2), having poor water hygiene (OR, 2.1;CI95, 1.1 to 3.9), having poor food hygiene (OR, 2.2; CI95, 1.1 to4.3), and using mosquito coils (OR, 1.8; CI95, 1.1 to 4.3) wereindependent determinants of K. pneumoniae nasopharyngeal car-riage. Poor water hygiene (OR, 1.8; CI95, 1.1 to 2.9), poor foodhygiene (OR, 1.8; CI95, 1.1 to 2.7) and being a female (OR, 1.9;CI95, 1.2 to 3.1) were independent determinants of other GNBcarriage.

This is the first population-based study of nasopharyngeal car-

riage of GNB in Indonesia. The carriage rate of GNB exceeded thatof S. pneumoniae in adults. Overall, 30% of the participants car-ried GNB. K. pneumoniae was the commonest GNB species car-ried, significantly higher among adults than among children. Fewpublications are available regarding nasopharyngeal carriage of K.pneumoniae or GNB. These bacteria are not considered commen-sals in the nasopharynxes of healthy individuals. K. pneumoniaeand other GNB throat carriage is found in specific conditions,such as preceding antibiotic therapy, underlying diseases, or longhospital stay (4). A study in Brazil, a high middle-income country(World Bank 2011) found a 9% carriage rate of GNB among 1,192children attending day care, with K. pneumoniae in only 1.4% of

TABLE 1 Characteristics of the study participants

Characteristic

No. (%) of subjects with thischaracteristic, unless specifiedotherwise

P valueaChildren Adults All subjects

GenderMale 118 (49) 94 (37) 212 (43) 0.01Female 125 (51) 159 (63) 284 (57)

Age (yr)Median 2.1 55Range 0.5–5 45–75

ResidenceUrban 180 (74) 186 (74) 366 (74) 0.9Suburban 63 (26) 67 (26) 130 (26)

Food hygieneGood 81 (33) 107 (42) 188 (38) 0.04Poor 162 (67) 146 (58) 308 (62)

Water hygieneGood 114 (47) 119 (47) 233 (47) 0.9Poor 129 (53) 134 (53) 263 (53)

Crowdingb

No 129 (56) 157 (66) 286 (61) 0.03Yes 100 (44) 80 (34) 180 (39)

Mosquito coil useNo 184 (76) 170 (67) 354 (73) 0.04Yes 59 (24) 83 (33) 142 (29)

Active smokingNo 243 (100) 200 (79) 443 (89) 0.01Yes 0 (0) 53 (21) 53 (11)

Passive smokingNo 97 (40) 106 (42) 203 (41) 0.7Yes 146 (60) 147 (58) 293 (59)

Contact with toddler(s)No 111 (46) 116 (46) 227 (46) 0.9Yes 132 (54) 137 (54) 269 (54)

Total no. 243 253 496a The P values are comparing the distributions of the variables for children versusadults.b Only 366 subjects responded to this question.

TABLE 2 Distribution of nasopharyngeal colonization among youngchildren and adults in Semarang, Java, Indonesia

Carriage typea

No. (%) of subjects with thiscarriage type

P valueChildren(n � 243)

Adults(n � 253)

All subjects(n � 496)

Noncarrier 107 (44) 157 (62) 264 (53) 0.000

Total carriersK. pneumoniae 16 (7) 38 (15) 54 (11) 0.004Other GNB 49 (20) 47 (19) 96 (19) 0.7S. pneumoniae 105 (43) 28 (11) 133 (27) 0.000

S. pneumoniae only 78 (32) 17 (7) 95 (19)K. pneumoniae only 5 (2) 25 (10) 30 (6)Other GNB only 22 (9) 39 (15) 61 (12)

S. pneumoniae andK. pneumoniae

4 (2) 7 (3) 11 (2) 0.000

S. pneumoniae and other GNB 20 (8) 2 (1) 22 (4)K. pneumoniae and other GNB 4 (2) 4 (2) 8 (2)S. pneumoniae, K. pneumoniae,

and other GNB3 (1) 2 (1) 5 (1)

a GNB, Gram-negative bacilli.

TABLE 3 Nasopharyngeal carriage of Gram-negative bacilli

Species

No. (%) of subjects carrying theindicated species

Children(n � 243)

Adults(n � 253)

All subjects(n � 496)

K. pneumoniae 16 (7) 38 (15) 54 (11)

Other GNBa 56 (23) 51 (20) 107 (21)Pseudomonas spp. 16 (7) 9 (4) 25 (5)Enterobacter spp. 11 (5) 10 (4) 21 (4)A. baumannii comp.b 11 (5) 3 (1) 14 (3)Citrobacter spp. 0 (0) 11 (4) 11 (2)Aeromonas spp. 6 (2) 3 (1) 9 (2)Pantoea spp. 6 (2) 3 (1) 9 (2)Serratia spp. 0 (0) 4 (2) 4 (1)Escherichia coli 0 (0) 3 (1) 3 (1)Stenotrophomonas maltophilia 1 (1) 1 (1) 2 (1)Other non-lactose-fermenting

bacillic5 (2) 4 (2) 9 (2)

Total carriage of GNB 72 (30) 89 (35) 161 (32)a GNB, Gram-negative bacilli.b Acinetobacter baumannii complex.c Other nonfermenting bacilli were Comamonas testosteroni, Leclercia adecarboxylata,Rhizobium radiobacter, Proteus vulgaris, Brucella melitensis, Shewanella algae, andSphingobacterium spiritivorum.

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the children (5). We showed that carriage of K. pneumoniae andother GNB in healthy individuals in Semarang, Indonesia, is muchmore common and related to poor hygiene, particularly water andfood hygiene.

K. pneumoniae and other GNB are present in the enteric mi-croflora which may serve as a source of water contamination par-ticularly in countries with poor sanitation (6). People who usecontaminated water could, thus, become colonized. Food prepa-ration using contaminated water may increase the risk of trans-mission to the nasopharynx. A study in neighboring Malaysiafound that 32% of street food contained K. pneumoniae (7).

Using mosquito coils was also a K. pneumoniae carriage de-terminant. It has been demonstrated that one mosquito coilreleases the same amount of particulate matter as 75 to 137cigarettes (8).

Carriage of K. pneumoniae and other GNB may play a role inthe pathogenesis of pneumonia in this part of the world (1), butthis hypothesis requires further study. Importantly, in this re-spect, almost all K. pneumoniae strains were found to have thewild-type susceptibility to common antimicrobial agents, sim-ilar to those found in CAP patients in Semarang, Indonesia(our unpublished observation). This finding confirms previousreports from the same community, where acquired resistancewas rarely found among enteric bacilli isolated from healthypeople (9, 10).

In conclusion, nasopharyngeal carriage of K. pneumoniae andother GNB is common among healthy people in Indonesia and iscaused in part by exposure to unsafe water and food.

ACKNOWLEDGMENTS

We thank medical students from the Faculty of Medicine of DiponegoroUniversity, Susilo Prihranto at Diponegoro University (Internal MedicineResidency Program), and technicians at the laboratory of Microbiologyand Infectious Diseases of Erasmus Medical Centre.

We declare that we have no conflicts of interest.

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