Mem Inst Oswaldo Cruz, Rio de Janeiro, 97(3) April 2002
Original Article

Pseudomonas aeruginosa: Study of Antibiotic Resistance and Molecular Typing in Hospital Infection Cases in a Neonatal Intensive Care Unit from Rio de Janeiro City, Brazil

MM Loureiro, BA de Moraes, VLF Mendonça, MRR Quadra*, GS Pinheiro*, MD Asensi

Laboratório de Enterobactérias, Departamento de Bacteriologia, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
*Hospital Maternidade Alexander Fleming II, Sistema Único de Saúde, Rio de Janeiro, RJ, Brasil

Page: 387-394
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This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized byu00a0Pseudomonas aeruginosau00a0sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period. The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively. In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia. We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains. All isolated strains were classified as multi-drug resistant. Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.

Pseudomonas aeruginosa is one of the most common pathogens involved in hospital infection (HI) causing opportunistic infections in humans, particularly among immunocompromised patients (Bert & Lambert-Zechovsky 1996, Kinoshita et al. 1997, Tsakris et al. 2000), and because of its ubiquitous nature, ability to survive in adverse conditions, and affinity for moist environments, remains a common pathogen in intensive care units (ICU) (Grundmann et al. 1995, Moolenaar et al. 2000)

The HI causes complication of medical care in ICUs, principally in neonatal intensive care units (NICU), due to the natural immunodeficiency of newborns, submission to invasive procedures of therapy and diagnostic, presence of several underlying risk conditions and indiscriminated antimicrobial use, and can causes significant morbidity and mortality (Sader et al. 1993, Kettner et al. 1995, Moolenaar et al. 2000). Mortality upon infected patients with P. aeruginosa sepsis was 46.7%, compared to only 21% for other bacteria in a study that collected blood stream isolates from nearly 50 medical center in the USA (Jones et al. 1997).

The worldwide emergence of multi-resistant bacterial strains is a growing concern, especially in HI cases caused by P. aeruginosa. Among nosocomial bacterial infections, those caused by P. aeruginosa are associated with highest mortality rate, and are difficult to eradicate from infected tissues or blood because those microorganisms are virulent and have a limited susceptibility to antimicrobials (Kettner et al. 1995, Harris et al. 1999).

The epidemiology of P. aeruginosa infections are usually studied by the analysis of phenotypic markers, including biotype, serovar, pyocin production, phage type, and antimicrobial susceptibility pattern (Pitt 1988, Kinoshita et al. 1997). Typing of strains is important for eradication of environmental sources as well as prevention of cross-infections and monitoring of antimicrobial therapy efficacy (Poh et al. 1992). Recently, nucleic acid-based methods have been used to assist identification of bacteria to subspecies level in epidemiological studies, providing a higher discriminatory power than phenotypic parameters (Severino et al. 1999)

Chromosomal DNA restriction analysis by pulsed field gel electrophoresis (PFGE) is considered worldwide the most powerful tool to perform hospital epidemiologic studies of P. aeruginosa because of its high discriminatory capacity. These technique facilitate the elucidation of transmission routes, genetic variability and phylogenetic distances between strains (Sader et al. 1993, Grundmann et al. 1995, Renders et al. 1996, Müller-Premru & Gubina 1999)

This study reports the demographics characteristics of hospitalized newborns seized by P. aeruginosa sepsis associated with HI, and antimicrobial resistance percentage, serotypes and PFGE patterns of the isolated strains in a NICU from a public maternity hospital in Rio de Janeiro city, Brazil.



Hospital and patients - Between July 1997 and July 1999, 32 P. aeruginosa strains were isolated from blood cultures of different NICU newborns involved in HI cases, at the Hospital Maternidade Alexander Fleming II (HMAF), Rio de Janeiro city, Brazil. It is a maternity hospital providing assistance and perinatal care including a neonatology intermediate care unity (NIU) with 40 beds and a NICU with 15 beds.

HI cases were defined according to Centers for Diseases Control and Prevention (CDC) (Garner et al. 1988). In general, infections that occurred after 48 h of permanence at the hospital were assumed to be hospital acquired.

Blood cultures, strains identification and susceptibility testing - 0.2 ml of venous blood obtained from newborns were drawn into bottles with 10 ml of Tripticase Soy Broth supplemented (Roche) and incubated to 37°C. After 24 h, the blood cultures were inoculated into Thioglycolate Broth (DIFCO) and plated on Blood Agar and Eosin-Methylene Blue Agar (EMB, DIFCO). The Thioglycolate Broth, and plates of Blood Agar and EMB Agar were incubated at 37°C during a period ranged from 18 to 24 h. When the blood cultures were negative after incubation of 24 h, the inoculation into broth and plates above cited were repeated during a week. Identification of P. aeruginosa strains were performed using the Crystal System of identification for fermenters and non-fermenters (BBL/Becton-Dickinson).

P. aeruginosa sepsis were defined by a single positive blood culture associated with appropriate clinical manifestations (one of the following clinical signs or symptoms: fever > 38°C, hypothermia < 36.5°, apnoea, bradycardia or tachycardia) according to CDC definitions (Garner et al. 1988)

The antimicrobial susceptibility test was carried out through of disk diffusion method according to National Committee for Clinical Laboratory Standards, NCCLS (1997) recommendations. Quality control was carried out using standard strains of Escherichia coli(ATCC 25922), P. aeruginosa (ATCC 27953) and Staphylococcus aureus (ATCC 25923). The following concentrations of antimicrobials drugs (CECON) were used: cephalothin (30 µg), cefoxitin (30 µg), ceftriaxone (30 µg), cefuroxime (30 µg), carbenicillin (100 µg), cefepime (30 µg), ceftazidime (30 µg), piperacillin/tazobactam (100/10 µg), ticarcillin/clavulanic acid (75/10 µg), imipenem (10 µg), gentamicin (10 µg), amikacin (30 µg), ciprofloxacin (5 µg), trimetho-prim/sulfamethoxazole (1.25/23.75 µg), chloramphenicol (30 µg), tetracycline (30 µg).

Serotyping method - An 18h Nutrient Agar culture (at 37°C) of each strain was used as antigen. One ml of sterile saline solution (0.85 % NaCl) was drawn into tubes containing the Nutrient Agar cultures and mixed to produce a suspension containing the bacterial growth. The O-group was identified by slide agglutination with P. aeruginosa Antisera Kit (Denka Seiken Co. Ltd., Tokyo, Japan), according to Homma (1982) and Liu et al. (1983).

PFGE method - The strains were submitted to chromosomal DNA extraction and processing according to the procedures previously described by Sader et al.(1994). Nucleic acids present in agarose plugs were digested with 10U of SpeI restriction enzyme at 37ºC during 20 h, and the electrophoresis procedure was carried out in 1% agarose gels and running buffer containing 0.5X TBE with a CHEF DR III pulsed field electrophoresis systems (Bio-Rad, California). Running conditions consisted of two ramps in sequence (ramp A consisted of an initial switch time of 0.5 sec, a final switch time of 25 sec, and a run time of 20 h; ramp B consisted of an initial switch time of 30 sec, a final switch time of 60 sec, and run time of 4 h). The voltage was 6V/cm for both ramps, and the temperature was kept constant at 13ºC. Fragments were stained with ethidium bromide and photographed. All agarose gels were run twice to verify the reproducibility of the tests.

Band patterns were analyzed using GelCompar II (Applied Maths, Belgium), without using internal markers. The similarities between fingerprints were determined by construction of a similarity matrix using the Dice's coefficient with 1.5% position tolerance and optimization of 1%, and a dendrogram generated using the UPGMA clustering algorithm. Definition of clonal structures of P. aeruginosa strains were made according to Tenover et al.(1995)

Epidemiological analysis of patients - For each patient, an epidemiological record with demographic and microbiologic data was done. These data were registered in an EXCEL 7.0 program (Microsoft) and analyzed later in Epi-Info program (version 6.04b; CDC, Atlanta, USA).

A control group of 70 newborns which did not acquire sepsis during the hospitalization in the NICU-HMAF from July 1997 to July 1998 was created. These control cases were randomly selected from a group of 720 patients that presented negative blood cultures, to evaluate the statistical significance of the demographics data. Most variables were compared using the odds ratios (ORs), 95% confidence intervals (CI95), chi-square (c2) and P values, except, mean age, mean of antimicrobial drugs used by patients before positive blood presentation and mean of antimicrobials days use before positive blood culture presentation, that were compared using the t-student test.



Table I shows the general demographic characteristics of the hospitalized newborns in NICU-HMAF, Rio de Janeiro city, Brazil, seized by P. aeruginosa sepsis and classified as HI cases. The mean age of these newborns was 12.5 days, and the major frequencies of HI were detected in male sex (59.4%) and vaginal delivery (81.2%). Twenty patients (62.5%) received antimicrobial drugs before positive blood cultures presentation with mean use of 3.6 antimicrobials per patient and mean of 11.8 days use. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight (VLBW), 40.6% had asphyxia and 31.2% presented Hyaline Membrane Disease (HMD).

The statistical results of the comparison between study and control groups (Table I) using OR, CI95, c2, P values and t-student test, showed statistical significance in mean of antimicrobial drugs used by patients before positive blood culture presentation (T = 3.00) and mean of antimicrobials days use before positive blood cultures presentation (T = 2.22).

A total of 11 different antimicrobials drugs (Table II) were used by 20 patients (62.5%) before positive blood cultures presentation, with major use frequency for ampicillin (18/20 patients-90%), amikacin (17/20-85%), cefotaxime (9/20-45%) and oxacillin (9/20-45%).

The antimicrobial resistance percentage of the isolated P. aeruginosa strains (Table III) demonstrated high antimicrobial resistance percentage (75 to 100% of resistance) to cephalotin, cefoxitin, ceftriaxone, cefuroxime, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline in two analyzed periods (July 1997 to July 1998 and August 1998 to July 1999); high antimicrobial resistance percentage to ceftazidime, cefepime, carbenicillin and ticarcillin/clavulanic acid during the first analyzed period and no resistant strain in the second; low antimicrobial resistance percentage (0 to 35% of resistance) to piperacillin/tazobactam in the first period and no resistant strain in second; low antimicrobial resistance percentage to gentamicin and amikacin (10.7% each) during the first period and were detected increase in antimicrobial resistance percentage in second (50 and 25%, respectively). In the first period there was no isolation of resistant strains to imipenem and ciprofloxacin, but in the second period an emergence of resistant strains was detected.

Fig. 1 shows seven distinct PFGE patterns (A-G) observed in the 32 P. aeruginosa strains, where the clone A was the most frequent (epidemic clone; 75% of the strains), while the patterns C and E presented two strains, and the patterns B, D, F and G only one strain, corresponding to 25% of the isolated strains in total.

The dendrogram generated by GelCompar II (Fig. 2), revealed that the seven clones presented low similarity percentage (< 80%) under stringency conditions above cited, indicating that these clones are not genetically related.

The serotyping results (Table IV) demonstrated four serotypes, with isolation predominance of the serotype E (29 strains; 90.7%), followed by serotypes B, D and I (one strain in each; 3.1%). The correspondence nomenclature of the serotypes B, D, E and I with the International Antigenic Typing Scheme (IATS) serotypes designation are respectively: O2/O5/O16, O9, O11 and O1 (Homma 1982, Liu et al. 1983).

The distribution of serotypes according to the chromosomal profiles (PFGE) and isolation periods is shown in Table IV. Strains with serotype O11 and chromosomal profile A (23 strains; 71.9%) were the more prevalent and have been isolated from July 1997 to June 1998. The others serotypes and chromosomal profiles were detected in small number distributed along the study period.

The distribution of chromosomal profiles and serotypes according to antimicrobial resistance profile (ARP) (Table V) showed 16 ARP including resistance ranged from 6 to 13 antimicrobials. Resistance profile 12 (28.1%) was the more frequent, followed by the profiles 13 and 15, with 12.6% of the isolates in each. Strains belonged to serotype O11 (90.7% of the strains) were distributed in 14 different ARPs (all, except ARPs 1 and 5), and demonstrated antimicrobial resistance varying from 6 to 13 antibiotics. The strains with PFGE pattern A (75% of the strains) were distributed in 10 different ARPs (1, 4, 6-9, 12-15), with antimicrobial resistance varying from 6 to 12 antimicrobials.



P. aeruginosa is the fourth most frequent pathogen isolated from nosocomial sepsis cases in the NICU-HMAF, accounting for 12.5% (32/255) of the infection cases, during the study period (data not shown). From the 32 sepsis cases detected in two years, 28 (87.5%) were detected in the first year of the study period. Sader (2000) related similar results notifying that P. aeruginosa was the third most frequent pathogen isolated from Gram-negative sepsis cases in a multi-centric study performed in Brazil, accounting 14.6% (125/855) of the cases. In another study on bloodstream infection cases in Latin America (Sader et al. 1999), 736 isolates were analyzed and P. aeruginosa was the fourth more frequent pathogen isolated in these cases, accounting 6.9% (51/736) of the isolates.

The newborns seized by P. aeruginosa sepsis in the HMAF, when compared with the control group presented as risk conditions for HI acquisition, a more elevated mean of antimicrobials drugs used before positive blood culture presentation (3.6 vs 2.1) and a major time use (11.8 daysvs 4.2 days) of these drugs, during the study period. These observations agree with other authors who described the extensive antimicrobial drugs use as predisposing risk factor to HI acquisition in NICUs, because these extensive use can select multi-drug resistant microorganisms (Jones et al. 1997, Leroyer et al. 1997, Cordero et al. 1999, Brodie et al. 2000).

Due the clinical picture suggestive of sepsis showed by infected newborns, a large number of patients (62.5%) received antimicrobials drugs before positive blood culture presentation with means use of 3.6 antimicrobials per patient and 11.8 days. In these cases, co-administration of ampicillin plus amikacin was the first empirical therapeutic scheme adopted in our institution, followed by oxacillin plus cefotaxime (second scheme). These results associated with high levels of antimicrobial resistance detected in the isolated strains, reinforce the idea that the empirical treatment adopted in hospital routine induced selective pressure of multi-drug resistant strains. Several authors described that prolonged use of antimicrobials before and during NICU stay can lead to selection of multi-resistant P. aeruginosa strains with different resistance forms, resulting in treatment failure, due selective pressure promoted by indiscriminated use of antimicrobials, principally broad-spectrum antimicrobials (Kettner et al. 1995, Bert & Lambert-Zechovsky 1996, Cailleaux et al. 1997, Cordero et al. 1999).

In relation to antimicrobial resistance, cephalothin, cefoxitin, ceftriaxone, cefuroxime, chloramphenicol, tetracycline and trimethoprim/sulfamethoxazole, should not be considered effective agents for P. aeruginosa sepsistreatment in our hospital unit, due the high resistance rates detected during the study period. Others authors detected high resistance levels to b-lactams and trimethoprim/ sulfamethoxazole in hospital units (Sader et al. 1993, Kettner et al. 1995).

We detected high resistance percentage for ceftazidime, cefepime, carbenicillin and ticarcillin/clavulanic acid during the first year of analysis and only sensible strains during the second year. These results indicate that resistance to these drugs were most associated with the epidemic strains isolated during the first year, in second year were no isolated these strains, and consequently these drugs can be newly considered effective in treatment of P. aeruginosa sepsis at HMAF routine. Others studies recommend the use of ceftazidime, carbenicillin and cefepime for P. aeruginosa sepsis treatment (Ismaeel 1993, Sader 2000). For ticarcillin/clavulanic acid, some studies related that this antimicrobial was little active against P. aeruginosa strains (Ismaeel 1993, Tassios et al. 1998).

In a multi-centric study, performed by Sader (2000) in Brazil, were detected that imipenem is the most active compound against P. aeruginosa infections followed by ciprofloxacin. These drugs are considered the most effective agents for treatment of P. aeruginosa sepsis at HMAF, but some resistant strains were isolated in the second period of analysis (August 1998 to July 1999). Several authors consider imipenem and ciprofloxacin as potent agents in treatment of infections caused by multi-resistant P. aeruginosa and alerted the emergence of mutants for these drugs in the last years, due the indiscriminate use of these antimicrobials (Jones et al. 1997, Sader 2000, Tsakris et al. 2000).

Gentamicin and amikacin are considered effective antimicrobial agents and are largely used in the HMAF routine, but were detected a low number of resistant strains at HMAF during two years of study. This fact reflect the importance of to control the use of these antimicrobials in the hospital unit, for preventing the emergence of aminoglycosides-resistant strains. In addition, Müller-Premru and Gubina (1999) recommend the start of the restriction of antimicrobials use, when aminoglycosides-resistant strains are detected. Gentamicin and amikacin are considered by some authors as a suitable aminogly-coside antibiotics against drug resistant P. aeruginosa (Kettner et al. 1995, Jones et al. 1997).

We detected low number of resistant strains to piperacillin/tazobactam in the first year of study and none during the second year, suggesting that these drugs can be considered as effective agents in P. aeruginosa sepsis therapy. Sader (2000) related that piperacillin/tazobactam is the third most active compound in a multi-centric study of antimicrobial resistance in Brazil. The use of piperacillin, as well as others b-lactams must be monitored, because these antibiotics induce selective pressure of b-lactamase producers strains, resulting in resistance development in the hospital unit (Cailleaux et al. 1997).

Different resistance mechanisms have been observed in P. aeruginosa, such as reduced permeability of antimicrobials through outer membrane, antimicrobial efflux mechanisms, changes in the lipopolysaccharide, modification of DNA gyrase protein and inactivation or modification of the antimicrobial structure through enzymes production (Bert & Lambert-Zechovsky 1996, Cailleaux et al. 1997, Jones et al. 1997, Esparragón et al. 1999, Tsakris et al. 2000). The 16 different ARPs observed in the strains isolated in this study presented resistance to antimicrobials include at least two different families of drugs with different action mechanisms, characterizing the analyzed strains as multi-drug resistant.

Several authors related that P. aeruginosa serotype O11 has been recognized as an important hospital problem in recent years, principally in epidemic situations, because this microorganism present multi-drug resistance with different resistance phenotypes (Pitt 1988, Kettner et al. 1995, Bert & Lambert-Zechovsky 1996, Kinoshita et al. 1997, Tassios et al. 1998, Esparragón et al. 1999, Müller-Premru & Gubina 1999). In this study, strains with serotype O11 showed 14 different ARPs with resistance varying from 6 to 13 antimicrobial drugs. This fact reflect the importance of controlling the emergence of strains with this serotype in NICUs.

The serotype O11 was the most prevalent (29/32- 90.7%), and in major number associated with PFGE pattern A (23/29-79.3%) but was also associated with the PFGE patterns B, E, F and G (20.7% in total). These associations demonstrated poor discriminatory power of the serotyping technique for epidemiologic studies application. The results showed by earlier studies, recommends serotyping (simple, cheap, fast, and present good reproducibility) as an initial screening procedure in epidemiological studies of P. aeruginosa (Renders et al. 1996, Bergmans et al. 1997). On the other hands, PFGE analysis included one strain serotype O9 into the PFGE profile of the epidemic strains (PFGE pattern A). A similar observation was described by Grundmann et al. (1995) in an study of 77 P. aeruginosa from unrelated sources in London, demonstrating genetically closely related strains from unrelated sources and different serotypes.

The strains with PFGE pattern A were considered as an epidemic clone restrict to the first year of analysis, this is probably due to elimination of these strains from hospital environment after reinforcement of HI prevention measures (measures of contact isolation such as: suitable handwashing, gloving and gown use for management of the newborns) and control (treatment of the infected newborns) in our hospital unit, initiated in May 1998 with the objective of to control a MRSA (methicillin-resistant S. aureus) outbreak, as previously described (Loureiro et al. 2000). After reinforcement of the prevention and control measures, no new outbreaks caused by MRSA and P. aeruginosa was observed in our hospital unit.

The epidemic strains (PFGE pattern A) showed 10 different ARPs, with resistance varying from 6 to 12 antimicrobials, that can be associated with selective pressure in hospital environment, principally in NICUs, resulting in resistance development. In addition, Harris et al. (1999) demonstrated that serial isolates with different antimicrobial profiles from individual patients represented the same strain, after typing though PFGE methodology, indicating that resistance to each class of antipseudomonal agents emerged sequentially after antibiotic exposure. Therefore, the multi-drug antibiogram is important to verify the emergence of resistance in the NICU, but resistance profile analysis is not a suitable epidemiological marker for detection of P. aeruginosa outbreaks,because changes in the antibiogram profile can occur, particularly during a long outbreak (Sader et al. 1993, Müller-Premru & Gubina 1999).

This study has epidemiological implication, because few studies exist on occurrence of sepsis cases caused by P. aeruginosastrains serotype O11 in NICU patients from Brazil and Latin America. Our observations emphasize the need for appropriate microbiological monitoring of P. aeruginosa strains implicated in hospital infections using both traditional and molecular methods, as well as, emergence monitoring of resistant strains to broad-spectrum antimicrobials in hospital environment, and the need to reinforce educational measures for prevention of nosocomial transmission of multi-drug resistant microorganisms.



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+Corresponding author. Fax: +55-21-2270.6565.
Received 23 May 2001
Accepted 4 February 2002
Financial suppor: CNPq and Papes (Fiocruz)

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