Species distribution and antimicrobial susceptibility of gram-negative aerobic bacteria in hospitalized cancer patients

Background Nosocomial infections pose significant threats to hospitalized patients, especially the immunocompromised ones, such as cancer patients. Methods This study examined the microbial spectrum of gram-negative bacteria in various infection sites in patients with leukemia and solid tumors. The antimicrobial resistance patterns of the isolated bacteria were studied. Results The most frequently isolated gram-negative bacteria were Klebsiella pneumonia (31.2%) followed by Escherichia coli (22.2%). We report the isolation and identification of a number of less-frequent gram negative bacteria (Chromobacterium violacum, Burkholderia cepacia, Kluyvera ascorbata, Stenotrophomonas maltophilia, Yersinia pseudotuberculosis, and Salmonella arizona). Most of the gram-negative isolates from Respiratory Tract Infections (RTI), Gastro-intestinal Tract Infections (GITI), Urinary Tract Infections (UTI), and Bloodstream Infections (BSI) were obtained from leukemic patients. All gram-negative isolates from Skin Infections (SI) were obtained from solid-tumor patients. In both leukemic and solid-tumor patients, gram-negative bacteria causing UTI were mainly Escherichia coli and Klebsiella pneumoniae, while gram-negative bacteria causing RTI were mainly Klebsiella pneumoniae. Escherichia coli was the main gram-negative pathogen causing BSI in solid-tumor patients and GITI in leukemic patients. Isolates of Escherichia coli, Klebsiella, Enterobacter, Pseudomonas, and Acinetobacter species were resistant to most antibiotics tested. There was significant imipenem -resistance in Acinetobacter (40.9%), Pseudomonas (40%), and Enterobacter (22.2%) species, and noticeable imipinem-resistance in Klebsiella (13.9%) and Escherichia coli (8%). Conclusion This is the first study to report the evolution of imipenem-resistant gram-negative strains in Egypt. Mortality rates were higher in cancer patients with nosocomial Pseudomonas infections than any other bacterial infections. Policies restricting antibiotic consumption should be implemented to avoid the evolution of newer generations of antibiotic resistant-pathogens.

Most of the previous studies with cancer patients have only focused on bloodstream infections. However, limited information is available regarding the spectrum and microbiology of these infections in sites other than the bloodstream, such as the urinary tract, respiratory tract, gastro-intestinal tract, and the skin. This is despite the fact that these infections are not rare.
Our group has previously studied the microbial spectrum and antibiotic resistance patterns of gram-positive bacteria in cancer patients [4]. In the present study, the microbial spectrum of gram-negative bacteria isolated from various infection sites in hospitalized cancer patients was examined. The spectrum studied was not limited to the most common gram-negative bacteria, but included lessfrequent gram negative bacteria as well. Both patients with hematologic malignancies (leukemic patients) and patients with solid tumors were included in the study. Thus, the resistance profile of the isolated gram-negative bacteria was examined. In addition, we detected mortality rates attributed to nosocomial infections caused by gramnegative isolates.

Patient specimens
Non-duplicate clinical specimens from urine, pus, blood, sputum, chest tube, Broncho-Alveolar Lavage (BAL), throat swabs, and skin infection (SI) swabs were collected from patients at the National Cancer Institute (NCI), Cairo, Egypt. The SI swabs were obtained from cellulitis, wound infections, and perirectal infections. For each specimen type, only non-duplicate isolates were taken into consideration (the first isolate per species per patient). Data collected on each patient consisted of demographic data including age, sex, admission date, hospitalization duration, ward, and sites of positive culture. Selection criteria included those patients who had no evidence of infection on admission, but developed signs of infection after, at least, two days of hospitalization. Ethical approval to perform the study was obtained from the Egyptian Ministry of Health and Population. All the included patients consented to the collection of specimens from them before the study was initiated.

Microbial identification
Gram-negative bacteria were identified using standard biochemical tests. We also used a Microscan Negative Identification panel Type 2 (NEG ID Type 2) (Dade Behring, West Sacramento, USA) to confirm the identification of gram-negative facultative bacilli. PID is an in vitro diagnostic product that uses fluorescence technology to detect bacterial growth or metabolic activity and thus can automatically identify gram-negative facultative bacilli to species level. The system is based on reactions obtained with 34 pre-dosed dried substrates which are incorporated into the test media in order to determine bacterial activity. The panel was reconstituted using a prompt inoculation system.

Biochemical tests
In each Microscan NEG ID Type 2 kit, several biochemical tests were performed. These included carbohydrate fermentation tests, carbon utilization tests, and specific tests such as Voges Proskauer (VP), Nitrate reduction (NIT), Indole test, Esculine hydrolysis, Urease test, Hydrogen Sulphide production test, Tryptophan deaminase test, Oxidation-Fermentation test, and Oxidase test.

Antimicrobial susceptibility testing
Both automated and manual methods were used to detect antimicrobial susceptibility pattern of the isolates. The Microscan Negative Break Point combo panel type 12 (NBPC 12) automated system was used for antimicrobial susceptibility testing of gram-negative isolates. A prompt inoculation system was used to inoculate the panels. Incubation and reading of the panels were performed in the Microscan Walk away System. Kirby-Bauer technique (disc diffusion method) was also used to confirm resistant gram-negative isolates. Discs of several antimicrobial disks (Oxoid ltd., Basin Stoke, Hants, England) were placed on the surface of Muller Hinton agar plates followed by incubation at 35°C. Reading of the plates was carried out after 24 h using transmitted light by looking carefully for any growth within the zone of inhibition. Appropriate control strains were used to ensure the validity of the results. Susceptibility patterns were noted.

Calculation of mortality rate
We only calculated attributable mortality which we defined as death within the hospital (or 28 days following discharge) [5,6], with signs or symptoms of acute infection (septic shock, multi-organ failure). Other deaths were considered deaths due to the underlying cancer and were excluded from calculations. In addition, patients with polymicrobial infections were excluded from the mortality rate calculation.

Serratia plymuthica
Other gramnegative species     (Table 4). It is noteworthy that no gram negative isolates were recovered from SI in leukemic patients ( Table 3).

Stenotrophomonas maltophilia
The antimicrobial resistance patterns of different gramnegative isolates from cancer patients were examined. Isolates of Escherichia coli, Klebsiella, Enterobacter, Pseudomona, and Acinetobacter species were resistant to most antibiotics tested including non-β-lactam antibiotics such as aminoglycosides (gentamicin) and quinolones (ciprofloxacin, levofloxacin). In addition, isolates exhibited simultaneous resistance to more than one non β-lactam drug (Tables 5 and 6).
Gram-negative isolates were highly resistant to cefotaxime and ceftazidime. Escherichia coli exhibited 66.2% and 55.7% resistance to Cefotaxime and Ceftazidime. The percentage resistance to cefotaxime and ceftazidime was also high in Klebsiella, Enterobacter, Pseudomonas, and Acitenobacter isolates (Tables 5 and 6). In addition, 70.2% of Pseudomonas species isolates exhibited simultaneous resistance to cefotaxime and ceftazidime. Other gram-negative species also exhibited similar high rates of resistance to both cefotaxime and ceftazidime (Table 7).
It should be noted that the use of Tazobactam (β-lactamase inhibitor) enhanced the activity of piperacillin against Acinetobacter, Pseudomonas, Enterobacter, Klebsiella, and Escherichia coli. Similarly, the use of Clavulanate restored    (Tables 5 and 6).

Discussion
The goal of this study was to characterize the microbial spectrum and antibiotic susceptibility profile of gramnegative bacteria in cancer patients. The most frequently isolated gram-negative bacteria from all clinical specimens were Klebsiella pneumonia followed by Escherichia coli (Table 1). Other studies reported that Escherichia coli and Klebsiella species were the most frequently isolated gramnegative pathogens in nosocomial infections from cancer and non-cancer patients [9,10]. Similarly, Bilal et al reported that Klebsiella pneumonia was the most common isolate in their hospital in Saudia Arabia [11].
Klebsiella pneumonia was the main isolated gram-negative bacteria from sputum and throat (Table 1). This is consistent with the work of Hoheisel et al in Germany who reported that Klebsiella species were among the most frequent gram-negative isolates from RTI [12]. Results in table 1 indicated that the main isolated gram-negative bacteria from blood were Escherichia coli and Pseudomonas species (Table 1). Other studies also reported Escherichia coli and Pseudomonas species to be among the most prevalent organisms causing bloodstream infections in USA [13].
In the present study, 18% of cancer patients developed SI (data not shown). This is consistent with other studies which reported significant surgical site infection rates in cancer treatment centers [14,15]. As shown in table 1, the most commonly isolated gram-negative bacteria from SI were Klebsiella pneumonia, Escherichia coli, and Pseudomonas aeruginosa. Vilar-Compte et al reported that Escherichia coli and Pseudomonas species were the most commonly isolated bacteria from surgical site infections at a cancer center in Mexico [15]. The main isolated organisms from urine were Escherichia coli and Klebsiella pneumonia (Table 1). This is reminiscent of the study by Espersen et al who demonstrated that UTI due to Escherichia coli were the most frequent infections in patients with myelomatosis [16].
In addition to the present study, the isolation of Burkholderia cepacia and other less-frequent gram-negative bacteria had been reported in other studies of nosocomial infections in cancer and non-cancer patients [17][18][19] ( Table 2). The low prevalence of Salmonella, Shigella, and Yersinia species reported in our study was not unusual in the realm of nosocomial infections in cancer patients. In his study on patients with acute leukemia, Gorschluter et al reported low frequency of enteric infections by Salmonella, Shigella, Yersinia, and Campylobacter [20].
As in tables 5 and 6, all gram-negative species examined were highly resistant to third-generation cephalosporins. Reports from Korea and other parts of the world indicted that nosocomial infections caused by Enterobacter, Citrobacter, and Serratia species were also resistant to third generation cephalosporins [21].
Isolates producing ESβL confer resistance to all β-lactam agents and to other classes of antimicrobial agents, such as amino glycosides and flouroquinolones, thus making it difficult to treat infections they produce [22]. Reports indicate a significant increase in ESβL-producers in recent years [23]. Invasive procedures, specifically catheterization, prolonged hospital stay and confinement in an oncology unit were found to be associated with ESβL production [24]. Ceftazidime and cefotaxime resistance are potential markers for the presence of Extended-Spectrum β lactamases (ESβL). Aztreonam resistance is also a potential marker for the presence of an ESβL-producing organism. Levels of resistance to aztereonam among gramnegative isolates (Tables 5 and 6) were higher than those reported few years ago in Egypt [25]. In addition, there were high percentages of cefotaxime/ceftazidime-resistant gram-negative isolates. All of this suggested ESβL produc-
Compared with second-generation quinolones (ciprofloxacin), the newest fluoroquinolones (levofloxacin, gatifloxacin) have enhanced activity against gram-positive bacteria with only a minimal decrease in activity against gram-negative bacteria [26]. However, the newer generation quinolones are still quite active against most Enterobacteriaceae (such as Enterobacter, Escherichia, Klebsiella) and non-fermentative gram-negative bacilli (such as Acinetobacter) with the exception of Pseudomonas aeruginosa [27]. Results in tables 5 and 6 demonstrated that whereas Klebsiella, Pseudomonas, and Acinetobacter were relatively more susceptible to newer quinolones than ciprofloxacin, Escherichia coli was more susceptible to ciprofloxacin.
Enterobacter was particularly susceptible to levofloxacin. Thus, an older or newer quinolone may be more active depending on the particular gram-negative species involved.
Previous studies in Egypt reported that resistance to imipenem was totally absent or very low [25,28]. A similar observation was made in a study in Turkey [29]. Other studies in Turkey, Italy, and France reported the presence of low levels of resistance to imipenem [30][31][32][33]. Acinetobacter and Pseudomonas species exhibited the highest resistance levels to imipenem. Enterobacter still exhibited considerable resistance to imipenem. Escherichia coli and Klebsiella exhibited lower, but still noticeable, resistance to imipenem. To our knowledge, this is the first study which reports significant levels of imipenem resistance in Egypt.
Escherichia coli isolates were highly resistant to ampicillin, ampicillin-sulbactam, aminoglycosides, and other antibiotics. El Kholy et al reported that Escherichia coli isolates from cancer patients in Egypt exhibited a low susceptibility pattern [25].
In a study conducted in Turkey, Acinetobacter baumannii was resistant to most antibiotics tested except meropenem, tobramycin, and imipenem [34]. Results in Table  6 showed that Acinetobacter species, as well as Pseudomonas species, were highly resistant to ceftazidime, aztereonam, piperacillin, and amino glycosides as was reported in other studies [35,36]. Some investigators noticed that geographic differences affected the resistance patterns of gram-negative bacteria such as Acinetobacter species [36].
In such a case, local surveillance will be important in order to determine the most adequate therapy for infections caused by such organisms. Nosocomial outbreaks of the gram-negative pathogen Enterobacter cloacae were previously reported [37,38]. Our study confirmed previous reports which indicated that Enterobacter species isolated from hospitalized cancer patients from Egypt were highly resistant to ceftazidime, cefotaxime and aztereonam [25].
The phenomenon of multi drug resistant pathogens had emerged in Egypt and worldwide in recent years due to excessive antibiotic misuse [25,39]. Thus, Pathogens resistant to cephalosporins (third or fourth generation), carbapenems, aminoglycosides, and fluoroquinolone had emerged [39]. This study showed that gram-negative isolates can be resistant to more than one non β-lactam drug.
As indicated in table 7, the mortality rate associated with Pseudomonas infections in cancer patients was 34.1%. Previous reports also indicated high mortality rates (22%-33%) associated with Pseudomonas and Escherichia coli infections in immuno-compromised patients [40,41]. Similarly, the mortality rate (16%) attributed to Acinetobacter species infections was not very different from mortality rates attributed to Acinetobacter species infections in other reports (14-20%) [42,43].
The high levels of antimicrobial resistance in gram-negative bacteria can be attributed to antibiotic misuse in Egypt. Policies on the control of antibiotic usage have to be enforced and implemented to avoid the evolution of newer generations of pathogens with higher resistance, not only to the older generation drugs, but also to the relatively new ones. In addition, the entire microbial spectrum in various infection sites, and not just bloodstream pathogens, should be taken into account when initiating empirical antibiotic therapy.