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CHAPTER ONE INTRODUCTION

1.1 Background and literature review

Klebsiella species are common causes of nosocomial infections such as urinary tract infections, pneumonia, wound infections and septicaemia. Klebsiella species have emerged as important pathogens capable of exhibiting resistance to many antibiotics including Penicillins and Aminoglycosides. Although Klebsiella species have generally been susceptible to third generation Cephalosporins there have been reports of resistance to these agents (1). Klebsiella pneumoniae is naturally resistant to Ampicillin and amoxicillin usually by the production of SHV-1 lactamase encoded on the chromosome or a transferable plasmid (2, 3). Because of this natural resistance, Klebsiella pneumoniae often colonizes the skin and mucosa of hospitalized patients treated with Ampicillin or similar drugs; it may go on to produce invasive infections and septicaemia. Most colonized patients are asymptomatic, but may act as sources of cross infection for others, with the outbreak strain usually being transferred on staff hands (4). Resistance to broad spectrum Cephalosporins has emerged in strains of members of the enterobacteriaceae family following frequent use of these drugs in hospital settings (5, 6).Endemic and epidemic nosocomial infections caused by extended spectrum lactamase (ESBL) producing Klebsiella pneumoniae cells represent a persistent problem in many parts of the world, especially in intensive care units (6, 7, 8, 9-12).Epidemic strains of cephalosporin-resistant Klebsiella pneumoniae have been associated with increased morbidity and mortality in hospitalized patients(6, 9). A study carried out on urinary pathogens isolated at Parirenyatwa hospital Harare (13) showed that 76.2% of Klebsiella isolates were susceptible to Nalidixic acid and 98% to ciprofloxacin and most isolates were resistant to Gentamicin (87.2 %) and Ampicillin (97.2%). Some studies have shown high resistance of Klebsiella species to Cephalosporins such as Cefaroxime and Ceftraixone (14). Emergence of Klebsiella strains resistant to Flouroquinolones has been reported in some countries (15).

1.1.1 EpidemiologyThe genus Klebsiella is a member of the Enterobacteriaceae family. Klebsiella species are ubiquitous in nature and can be found in the natural environment (e.g., water and soil) and on mucosal surfaces of mammals (16). Common sites of colonization in healthy humans are the gastrointestinal tract, eyes, respiratory tract, and genitourinary tract (16).

Klebsiella pneumoniae has emerged as an important cause of hospital-acquired infections, especially among patients in the neonatal intensive-care unit (NICU) and mortality rates can be as high as 70% (17). Over the last two decades, the incidence of infections caused by multidrug-resistant Klebsiella strains has increased.

Extended spectrum -lactamase enzymes were first described in Klebsiella pneumoniae and Serratia marcescens isolates in 1983 in Europe and in Klebsiella pneumoniae and Escherichia coli isolates in 1989 in the United States (18). Since then, there has been a marked increase in the incidence of bacteria that produce ESBL enzymes. In the United States, the proportion of Klebsiella pneumoniae strains resistant to Ceftazidime increased from 1.5% in 1987 to 3.6% in 1991, and by 1993 as many as 20% of strains were resistant to Ceftazidime in some teaching hospitals (19,20 ). Of 824Klebsiella pneumoniae strains isolated from 15 hospitals in New York City during 1999, 34% expressed ESBL enzymes (21).

1.1.2 Virulence factors and pathogenesis

A number of virulence factors have been suggested in K. pneumoniae, including the prominent polysaccharide capsule expressed by the vast majority of clinical isolates as well as different adhesins, lipo polysaccharide (LPS) and iron-scavenging proteins (16).Knowledge of the exact role of individual K. pneumoniae virulence factors in different types of infection is, however, sparse and even less is known of the factors involved in the ability of the bacteria to colonize and reside in the GI tract of the host. Such knowledge is important in the search for new strategies to treat and prevent K. pneumoniae infections.

The vast majority of clinical K. pneumoniae isolates express a prominent polysaccharide capsule that covers the bacterial cells. The capsule is generally considered to be a major virulence factor in K. pneumoniae, especially in systemic infections, whereas its role in serum-poor host environments is less clear (16). Extracellular capsules are essential to virulence; the capsular material forms thick bundles of fibrillous structures that cover the bacterial surface in massive layers (16). This protects the bacterium from phagocytosis by polymorphonuclear granulocytes and prevents killing by bactericidal serum factors via the complement-mediated cascade. Currently, about 80 different capsular (K) antigens are known. Although Klebsiella capsular polysaccharide (CPS) has generally been thought to mediate virulence, it has been shown more recently that the mannose content of the CPS confers the degree of virulence. For example, strains that contain repetitive sequences of mannose-alpha-2 or alpha 3-mannose, L-rhamnose-alpha-2 or 3-L-rhamnose are of lower virulence. These mannose sequences are recognized by the surface lectin of macrophages and the organism is more efficiently ingested and killed by opsonin-independent phagocytosis. (22)

In addition to the capsule, there are about five somatic or O antigens, fimbrial and nonfimbrial adhesins, which serve as virulence factors. The fimbriae or pili are nonflagellar, filamentous projections on the bacterial surface that mediate attachment of the organism to respiratory, gastrointestinal, and urinary tract mucosal cells. Most clinical K. pneumoniae isolates are able to produce the two fimbrial adhesins, type 1 fimbriae and type 3 fimbriae. Type 1 fimbriae mediate adhesion to mannose-containing structures on host cells and extracellular matrix and are present in many species of Enterobacteriaceae.Additional virulence determinants for Klebsiella species include the ability of the organism to scavenge iron from the surrounding medium using secreted siderophores, that is, enterochelin and aerobactin. These are high-affinity, low molecular weight iron chelators that competitively take up iron bound to host proteins (23).

1.1.3 Molecular mechanisms of resistance

ESBL are plasmid-mediated enzymes that hydrolyze oxyimino- lactam agents such as third-generation Cephalosporins and Aztreonam (24). These plasmids also carry resistance genes to other antibiotics including aminoglycosides, Chloramphenicol, sulphonamides, Trimethoprim, and tetracycline. Thus, Gram negative bacilli containing these plasmids are multidrug-resistant (25). Furthermore, these plasmids are mobile genetic elements and can be transmitted between Gram negative bacilli of different species in vivo (26). During a 30-month outbreak of ESBL-producing K. oxytoca in an NICU, the plasmid from K. oxytoca spread to K. pneumoniae, E. coli, Enterobacter cloacae, and Citrobacter freundii (27).

Over 100 different ESBL enzymes have been identified, each with a preferential substrate. Thus, an ESBL-producing isolate may be resistant to Ceftazidime, but susceptible to Cefotaxime. As a result, ESBL-producing isolates may not be detected if susceptibility testing is limited to a single third-generation cephalosporin. The National Committee for Clinical Laboratory Standards (NCCLS) recommends routine screening for ESBL activity in E. coli, K. pneumoniae, and K. oxytoca isolates by determining susceptibility to several Cephalosporins including Cefpodoxime, Cefotaxime, Ceftriaxone, and Ceftazidime (28,29 ).

If an isolate is resistant to any one of these agents, that is, minimum inhibitory concentration (MIC) 2g/ml, confirmatory tests for an ESBL enzyme are performed by demonstrating increased susceptibility to Cefotaxime or Ceftazidime in the presence of Clavulanic acid, as Clavulanic acid inhibits ESBL enzymes and lowers the MIC of the Cephalosporins. However bacteria with ESBL-containing plasmids remain susceptible to the Carbapenems, that is, Meropenem and Imipenem, and Cephamycins such as Cefoxitin and Cefotetan.1.1.4 Risk Factors for Acquisition of ESBL-producing Pathogens

Epidemiological studies suggest that the increasingly widespread use of third-generation Cephalosporins is a major risk factor that has contributed to the emergence of ESBL-producing K. Pneumoniae (6, 12, and 30). Several additional risk factors for colonization and infection with ESBL-producing organisms have been reported and include: arterial and central venous catheterization, gastrointestinal tract colonization with ESBL-producing organisms, prolonged length of stay in an intensive-care unit, low birth weight in preterm infants, prior antibiotic use, and mechanical ventilation (31, 32, and 33). Carriage of this organism increases dramatically among hospitalized patients, as colonization rates increase in direct proportion to the length of stay (27).

Outbreaks of ESBL-producing organisms have been described. Asymptomatic patients colonized with ESBL-producing K. pneumoniae can serve as reservoirs for this pathogen with subsequent patient-to-patient spread via the hands of health-care workers. In addition, contaminated patient-care items and artificial fingernails worn by health-care workers have been implicated in transmission (34, 35, 36, and 37). Most studies have demonstrated a poor adherence to infection control policies as an important factor. Outbreaks of ESBL-producing K. pneumoniae in NICUs have been notable for high attack rates and large numbers of colonized infants (37).The neonates at greatest risk for colonization are those with a longer length of stay, a lower estimated gestational age and or a lower birth weight (27).

1.1.5 Clinical presentations

The importance of Gram negative bacilli as major causes of hospital-acquired infections in NICU patients has been well documented. K. pneumoniae can cause early-onset and, more commonly, late-onset sepsis, conjunctivitis, hospital-acquired pneumonia, urinary tract infections (UTIs), and surgical site infections (16, 38). Approximately 4% of episodes of late-onset sepsis in very low birth infants and 6% of overall infections in the neonatal ICU population are caused by K. Pneumoniae (39, 40). According to the most recent National Nosocomial Infection Surveillance System data, K. pneumoniae has been noted to cause 2.9% of bloodstream infections, 2.9% of eye, ear, nose and throat infections, 9.8% of GI tract infections, 5.7% of pneumonia, and 6.3% of surgical site infections in the NICU (41). As demonstrated by the CDC/National Association of Children's Hospitals and Related Institutions (NACHRI) point-prevalence survey conducted in 1999, K. pneumoniae caused 1.7% of bloodstream infections, 20% of respiratory tract infections, 8.3% of UTIs, and 5.6% of other infections in patients in NICUs (40).The present study is designed to determine the prevalence of Klebsiella species in clinical specimens at Parirenyatwa hospital and to determine the antimicrobial susceptibility patterns of the isolates. 1.2 Statement of the problem

Considering the ubiquitous nature of Klebsiella species and their intrinsic resistance to a wide variety of antimicrobial agents the organisms are able to survive within a hospital setting. Endemic and epidemic nosocomial infections caused by extended spectrum lactamase (ESBL) producing Klebsiella pneumoniae cells represent a persistent problem in many parts of the world, especially in intensive care units (6, 7, 8, 9-12). Klebsiella species are successful opportunistic pathogens thus their prevalence and antimicrobial susceptibility patterns need constant evaluation so as to reduce further health risks, recurrence and continual spread of infection within hospital settings.

1.3 Hypothesis

Null Hypothesis (Ho)

The prevalence of Klebsiella species in clinical specimens at Parirenyatwa is 10%

Alternative Hypothesis (Ha)

The prevalence of Klebsiella species in clinical specimens at Parirenyatwa is greater than 10%

1.3 Specific objectives

To determine the prevalence of Klebsiella species in clinical specimens at Parirenyatwa To determine the antimicrobial susceptibility patterns of the isolates.CHAPTER TWO MATERIALS AND METHODS

2.1 MaterialsLatex examination gloves

Bunsen burner

Light microscope

Lens tissue

Diamond marker

Immersion oil

76 26mm glass slides

Slide rack

Crystal violet

Lugols iodine

Acetone

Carbol fuschinIncubator

Gram negative antibiotic discs: Tetracycline (25g), Kanamycin (30g), Chloramphenicol (25 g), Ampicillin (25 g), Streptomycin (10g), Cotrimoxazole (25g), Amikacin (10g), Gentamicin (30g) and Ceftazidime (30g).Distilled water

Sulphuric acid

Barium chloride

Inoculating wire loop

MacConkey agar

Muller Hinton agar

Lysine iron agar

M.RVP (Methyl red Voges- Proskauer) broth

Simmons citrate agar

Kovacs reagent

Peptone water

Petri dishesMighty marker

Bijou bottles

Long test tubes

Khan tubes

Cotton wool

Meter rule

Tongs Physiological saline2.2 Methods2.2.1 Study site

The study was conducted at Parirenyatwa Central Hospital which is one of the countrys largest referral hospitals where patients are received from local clinics, district and provincial hospitals.

2.2.2 Study design

A cross sectional study was carried out on clinical specimens (urines and pus swabs) of patients at Parirenyatwa Central Hospital. Isolates identified as Klebsiella species were tested for antimicrobial susceptibility patterns2.2.3 Study subjects

The study was carried out on urine and pus swab specimens sent to Parirenyatwa Hospital Public Health Microbiology laboratory for microscopy, culture and sensitivity. The study was performed during the periods April 2010 to June 2010.

2.2.4 Sample size

A total of 150 specimens were analysed during the study. Statistically this was sufficient enough to determine the prevalence and antimicrobial susceptibility patterns of Klebsiella species. (Appendix I).

2.2.5 Inclusion criteria

Inclusion into the study required specimens sent to Parirenyatwa Hospital Public Health Microbiology laboratory accompanied by laboratory request form with patient demographic data. Only specimens that reached the laboratory within twenty four hours of their collection were included2.2.6 Exclusion criteria

Specimens delivered to the laboratory after 24 hours of their collection were excluded. Specimens which were not accompanied by patient details and clinical data were not enrolled into the study.2.2.7 ProcedurePermission to carry out the study was sought from the Clinical Director of Parirenyatwa Group of Hospitals and from the Chairman of the Joint Research Ethics Committee. After being granted, the study commenced in mid April 2010 and concluded in mid June 2010.Samples which were included in the study were allocated study numbers. The age, sex and clinical data for each specimen were also recorded. All identifying details were removed from specimens after allocation of a study number and results were correlated after completion of all assays.

2.2.7.1 Culturing of specimensPus swab specimens were inoculated on MacConkey agar plates using the isolation technique. The inoculated plates were incubated aerobically at 37C overnight, after which characteristic colonies were observed.Using a standard graduated inoculating wire loop, urine specimens were cultured on MacConkey agar plates using the isolation technique. The inoculated plates were incubated aerobically at 37C overnight, after which characteristic colonies were observed.

2.2.7.2 Identification of isolatesAfter observing visible growth on the culture plates the following procedures were performed: Isolates were checked for lactose fermentation on MacConkey agar plates, typical lactose fermenting colonies were observed as pink colonies. Colonies were checked for mucoid appearance which is a typical feature of Klebsiella species.

Colonies were emulsified with saline on a glass slide to make a smear.

Smears were stained using the Gram stain technique (Appendix II) and were observed under the light microscope at the 100x objective lens under immersion oil to identify the respective gram reactions. Isolates identified as lactose fermenting, gram negative rods and suspected to be Klebsiella were then sub cultured on fresh MacConkey agar plates to obtain pure cultures for further identification based on biochemical reactions (Appendix III) of the pure isolates.Table 1.Biochemical tests used to differentiate Klebsiella pneumoniae subspecies SpeciesVoges-ProskauerLactose fermentationUreaseCitrateMalonate utilizationLysine decarboxylase

K.p pneumoniae - + + + + +

K.p aerogenes + + + + + +

K.p ozaenae - - -

K.p rhinoscleromatis - - - - + -

Key: K.p = Klebsiella pneumoniae

2.2.7.3 Antimicrobial Susceptibility Testing

The Kirby-Bauer disk diffusion test, which conforms to the recommended standard of the National Committee for Clinical Laboratory Standards (NCCLS), was used in the study. Briefly, a small inoculum of each pure bacterial isolate was emulsified in 3ml sterile normal saline in Bijou bottles and the density of the solution was compared to a barium chloride standard (0.5 McFarland). The standardised solution of bacterial cultures was poured onto Muller Hinton agar plates to evenly inoculate the culture media. The excess bacterial suspension was discarded from the agar and the inoculated plates were left to dry. Thereafter, antibiotic disks with the following drug contentsAmpicillin (25g), Amikacin (10g), Chloramphenicol (25g), Cotrimoxazole (25g), Gentamicin (30g), Kanamycin (30g), Streptomycin (10g) and Tetracycline (25g) were placed on the plates, spacing them well to prevent overlapping of inhibition zones. The plates were incubated aerobically at 37C overnight and the inhibition zone diameters were then compared with recorded diameters of the control organism, E. coli ATCC 25922 to determine susceptibility or resistance.2.2.7.4 Data analysis

Data was captured using Microsoft excel and all statistical analysis performed by Epi info. The hypothesis was tested using the standard Z test at 5% level of significance whether it is true. The prevalence of Klebsiella species in clinical specimens was calculated at 5% level of significance and the antimicrobial susceptibility patterns were determined through comparison with standard patterns of control organisms. CHAPTER THREE RESULTS AND ANALYSIS

During the study, 150 specimens were processed for Klebsiella positivity; of the 150 specimens only 18 specimens were positive for Klebsiella pneumoniae. Of the positive specimens 11(61%) were females and 7(39%) were males (Fig 1). The patients age ranged from 4 years to 85 years with a mean of 36 years and a median of 30 years. The calculated prevalence of the isolates was 12% (Table 2).The discrete variables were analysed using the standard normal Z test (Appendix IV). At 5% level of significance, statistically there was no sufficient evidence to reject the null hypothesis.Figure 1. Pie chart for patients sex distribution in positive specimens.

Table 2.Distribution of isolates in clinical specimens

TYPE OF SPECIMENNUMBER OF SAMPLESNUMBER ISOLATED% PREVALENCE

Urine901118.33

Swab6077.78

Total1501812

Table 3 shows the antimicrobial susceptibility results exhibited by Klebsiella pneumoniae isolates. Of the 18 isolates tested, most strains showed varied susceptibilities to the Aminoglycosides (Gentamicin and Streptomycin). Over 85% of the isolates were resistant to the sulphonamide Cotrimoxazole and 22% of the isolates were sensitive to Kanamycin, Chloramphenicol, Amikacin and Gentamicin. Over 80% of the isolates were resistant to two antibiotics or more. However, all isolates showed total resistance (100%) to Ampicillin. Table 3 Antimicrobial susceptibility patterns of Klebsiella pneumoniae strains isolated from various clinical specimens.Antibiotic Sensitive Intermediate Resistant

Frequency Percentage Frequency Percentage frequencyPercentage

Chloramphenicol 422.22211.111266.67

Ampicillin000018100

Tetracycline211.11211.111477.78

Streptomycin 00316.671583.3

Cotrimoxazole 211.11001688.89

Kanamycin422.22422.221055.56

Amikacin422.22527.78950.00

Gentamicin 422.22211.111266.67

All isolates demonstrated total resistance to Ampicillin. Based on the susceptibility to other aminoglycosides (Gentamicin, Amikacin and Streptomycin), Tetracycline, Cotrimoxazole, Kanamycin and Chloramphenicol, 7 Antibiotypes (A1A7) were defined for the 18 Klebsiella pneumoniae isolates (Table 4). Most of the isolates sixteen were resistant to three or more antibiotics. The predominant antibiotype (A1) constituted sixteen (88.89%) of the isolates while the least patterns (A6 and A7) were exhibited by six (33.33%) isolates respectively.Table 4. Antibiotypes of Klebsiella Pneumoniae isolates from various specimens.Antibiotypes Frequency Percentage

A1 1688.89

A21372.22

A31055.56

A41266.67

A51055.56

A6633.33

A7633.33

A1= Resistance to Ampicillin and Cotrimoxazole. A2= Resistance to Ampicillin, Cotrimoxazole and Streptomycin. A3= Resistance to Ampicillin, Tetracycline and Chloramphenicol. A4= Resistance to Ampicillin, Cotrimoxazole, Tetracycline and Streptomycin. A5= Resistance to Ampicillin, Cotrimoxazole, Tetracycline, Streptomycin and Chloramphenicol. A6= Resistance to Ampicillin, Gentamicin, Amikacin and Kanamycin.A7= Resistance to Ampicillin, Streptomycin, Cotrimoxazole, Tetracycline, Chloramphenicol and Kanamycin.Klebsiella pneumoniae strains isolated from pus swab specimens demonstrated total resistance to Ampicillin and Cotrimoxazole, whilst only 28.57% of the isolates were sensitive to Amikacin (Figure 2). All the isolates showed zero sensitivity to all the antibiotics except Amikacin. Fig 2.Antimicrobial susceptibility patterns of Klebsiella pneumoniae strains isolated from pus swab specimens.

Fig 3. Antimicrobial susceptibility patterns of Klebsiella pneumoniae strains isolated from urine specimens.

CHAPTER FOUR DISCUSSION AND CONCLUSION4.0 DiscussionKlebsiella pneumoniae accounts for a substantial degree of nosocomial infections (1, 2) and the increasing tendency for nosocomial pathogens to acquire new antibiotic resistance traits poses a problem in the control of infections. This feature reveals the need for an updated antibiotic susceptibility pattern for the effective management of infections caused by Klebsiella pneumoniae. The present study reports on the antimicrobial susceptibility patterns of Klebsiella pneumoniae isolates and the prevalence of the isolates at the study site. The findings revealed that Klebsiella pneumoniae isolates showed marked resistance to Ampicillin, Cotrimoxazole Tetracycline, Gentamicin and Chloramphenicol. Total resistance was exhibited on Ampicillin (100%), (88.89%) resistance to the sulfonamide Cotrimoxazole followed by (77.78%) resistance to Tetracycline. Gentamicin and Chloramphenicol both exhibited (66.67%) resistance from the isolates. However a few isolates were sensitive to Aminoglycosides and most of the isolates were resistant. The isolates showed extremely low levels of susceptibility (22.22%) to Gentamicin, Chloramphenicol, Amikacin and Kanamycin which was recorded as the highest degree of susceptibility indicating increased resistance of the isolates. In the same vain of analysis Cotrimoxazole and Tetracycline exhibited (11.11%) susceptibility whereas none of the isolates showed susceptibility to Ampicillin and Streptomycin. The overwhelming increased resistance of Klebsiella pneumoniae isolates may be attributed to the production of ESBL. On the other hand speculations could be raised in that the non determination of ESBL production could be a contributing factor to this unusual susceptibility since the production of ESBL has been reported to cause hidden resistance to broad spectrum Cephalosporins (14).

The extreme resistance to Gentamicin and Ampicillin were also observed corroborating a previous study carried on urinary pathogens at Parirenyatwa Hospital (13), with a slight increase in resistance to Ampicillin from (92.7%) to total resistance (100%). In addition Cotrimoxazole and Tetracycline also exhibited increased resistance from the isolates, this outcome could be linked to the fact that Cotrimoxazole, Tetracycline, Gentamicin and Ampicillin are commonly used in the environment under study to treat other infections, and are highly abused thus putting selective pressure on the antibiotics, hence the emergence of resistance to the antimicrobial drugs. Results of this study revealed seven distinct resistance patterns (Antibiotypes), of which the most prevalent exhibited resistance to Ampicillin and Cotrimoxazole accounting for 88.89% of the isolates (Table 4). The least patterns A6 and A7 were exhibited by six (33.33%) isolates respectively. All the Antibiotypes shared resistance to Ampicillin and this suggests that the drug may not be useful in treatment of Klebsiella pneumoniae infections in the area studded. An interesting feature of the study was that all Klebsiella pneumoniae isolates were resistant to three or more antibiotics. In addition five (27.78%) of the isolates exhibited total resistance to all the antibiotics used in the study. The multiple resistances to these drugs could be attributed to the fact that these antibiotics are highly abused due to constant and indiscriminate usage (13). Also Klebsiella pneumoniae strains are known to be an important source of transferable antibiotic resistance among gram negative bacilli. (5-8, 30). Consequently, the occurrence of multiple resistant Klebsiella pneumoniae strains might pose a potential problem in the treatment and management of infections in the study area.4.1 Limitations

Only 150 samples were obtained for the study which was not sufficient enough to draw feasible conclusions. Due to shortage of time the samples lacked randomization hence all samples available were used for the study. The amount of resources was limited and some procedures could not be undertaken, for example: the unavailability of third generation Cephalosporins (Ceftazidime, Clavulanate and Cefuroxime) which were required for demonstration of ESBL production by the isolates. Blood culture specimens and sputum specimens suspected of pneumoniae which were crucial for the study were unavailable, thus were left out.4.2 RecommendationsIf the study could be performed with adequate amounts of resources and over a long period of time fruitful results can be obtained. The isolates obtained should be Sero-typed and demonstration of ESBL production should be done so as determine the prevalence and antimicrobial susceptibility patterns of species specific isolates.4.3 Conclusion

Results from the study revealed that Klebsiella pneumoniae isolates were resistant to three or more antibiotics, with some exhibiting total resistance to all antibiotics and a few isolates exhibited sensitivity. Klebsiella pneumoniae was identified to be an important cause of urinary tract infections especially amongst females. This outcome reveals the need for an updated antibiotic susceptibility pattern for the effective treatment and management of infections caused by Klebsiella pneumoniae.ANTIBIOTIC

PERCENTAGE

PERCENTAGE

ANTIBIOTIC

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