Epidemiological analysis of bacterial strains involved in hospital infection in a University Hospital from Brazil

Análise epidemiológica de cepas bacterianas envolvidas em infecção hospitalar em um Hospital Universitário no Brasil

EPIDEMIOLOGICAL ANALYSIS OF BACTERIAL STRAINS INVOLVED IN HOSPITAL INFECTION IN A UNIVERSITY HOSPITAL FROM BRAZIL

Bianca Aguiar DE MORAES(¹), Cristiane Andrade Nery CRAVO(¹), Marcio Martins LOUREIRO(¹), Claude André SOLARI(²) & Marise Dutra ASENSI(¹)

SUMMARY

Hospital infections cause an increase in morbidity and mortality of hospitalized patients with significant rise in hospital costs. The aim of this work was an epidemiological analysis of hospital infection cases occurred in a public University Hospital in Rio de Janeiro. Hence, 238 strains were isolated from 14 different clinical materials of 166 patients hospitalized in the period between August 1995 and July 1997. The average age of the patients was 33.4 years, 72.9% used antimicrobials before having a positive culture. The most common risk conditions were surgery (19.3%), positive HIV or AIDS (18.1%) and lung disease (16.9%). 24 different bacterial species were identified, S. aureus (21%) and P. aeruginosa (18.5%) were predominant. Among 50 S. aureus isolated strains 36% were classified as MRSA (Methicillin Resistant S. aureus). The Gram negative bacteria presented high resistance to aminoglycosides and cephalosporins. A diarrhea outbreak, detected in high-risk neonatology ward, was caused by Salmonella serovar Infantis strain, with high antimicrobial resistance and a plasmid of high molecular weight (98Mda) containing virulence genes and positive for R factor.

KEYWORDS: Hospital infection; Antimicrobial resistance; Salmonella outbreak

INTRODUCTION

Hospital Infections (HI) are a public health problem, mainly, because they increase morbidity and mortality of hospitalized patients, time of hospital stay and costs of treatment (JARVIS, 1987).

In developed countries HI ranges from 5 to 10% of hospitalizations (BERGOGNE-BÉRÉZIN, 1995). It is estimated that in the United States from 25,000 to 100,000 deaths are caused directly by HI, with correspondent costs of over 7.5 billion dollars a year (WENZEL, 1994; SMITH & DOEBBELING, 1996).

Some risk conditions, which are likely to cause HI, have been reported. Some of them are: rise in the number of surgeries, use of catheters, mechanical ventilation, immunosuppressors and new bacterial strains resistant to antimicrobials (AL ORAINEY et al., 1989; SARTOR et al., 1995; KOELEMAN et al., 1997).

The microorganisms related to HI have varied in the last five decades, due to environmental factors and selective pressure of antimicrobial use. Thus, in the 50s the first outbreaks of S. aureus resistant to penicillin happened between 1960-1980, S. aureus resistant to oxacillin emerged as well as the Gram negative fermenters, which produce b-lactamases. After 1980 there were reports of Gram negative non-fermenters, Candida sp. and some types of virus (JARVIS, 1987, AL-ORAINEY et al., 1989, COTTON et al., 1989, BERGOGNE-BÉRÉZIN, 1995; SARTOR et al., 1995, MONNET et al., 1997).

This study reports the frequency of the microorganisms associated with HI in a public University Hospital in Rio de Janeiro, Brazil. It emphasizes the distribution of the microorganisms in several wards, anatomic sites, resistance to antimicrobials and epidemiological data of the patients infected.

MATERIAL AND METHODS

Description of the Hospital and Population

Bacterial strains were isolated from patients in different hospital wards of the Hospital Universitário Gafrée & Guinle (HUGG) – Universidade do Rio de Janeiro (UNI-RIO), Brazil. This hospital provides secondary and tertiary assistance to patients of different ages, from several places in Rio de Janeiro City and State. The data collected on each infection included the date, site of infection and patient demographic characteristics.

Bacterial Strains and Susceptibility Testing

A total of 238 bacteria strains isolated from 14 different anatomic sites (Table 2) were obtained from patients with infection. The criteria for defining nosocomial infection followed the National Nosocomial Infection Surveillance System (NNISS) according recomendations of Ministry of Health-Brazil, 1994. NNISS defines a nosocomial infection as any infection that is not present or incubating at the time the patient is admitted to the hospital, so was considered IH as being any infection occurrence after 48h of hospitalization. Each patient´s record was analysed criteriously, and based on clinical aspects, laboratorial data, type of infection and clinical specimen, the strain was classified as representative of infection or not. Surgical site infection were classified as infection occurrence within 30 days of the operation. Neonates infections were classified as any infection acquired after birth. All ConS strains were reported as the cause of a bloodstream infection only if the patient had fever, chills or hypotension and had no clinical evidence of sepsis at another site. The culture and identification followed the rules of BALOWS et al. (1991). The Gram-negative identification was completed by the Crystal System of identification for fermenters and non-fermenters (BBL/Becton-Dickinson). The antimicrobial susceptibility test was carried out through disk diffusion method in agar-disk of CECON, complying with NCCLS (National Committee for Clinical Laboratory Standards) recommendations (1995). Quality control was carried out using standard strains of Escherichia coli (ATCC 25922), P. aeruginosa (ATCC 27953) and S. aureus (ATCC 25923).

Plasmid Profile Analysis

Plasmid Profile analysis was performed through the alkaline lysis method of BIRNBOIM & DOLY (1979), modified by SAMBROOK et al. (1989). Samples of 15ml plasmid preparation were loaded into wells with 0.8% agarose gel of 7.5 cm length and run at 40V for 2 hours on a horizontal electrophoresis apparatus with a constant power source. Gels were stained with ethidium bromide and visualized on an UV transilluminator. Photographs were taken with a Polaroid Type 667 film. The plasmidial DNA of Escherichia coli R861 and V517 (THRELLFALL et al., 1986; MACRINA et al., 1978) were included as molecular controls.

Determination of the presence of R factors

Cultures of Salmonella serovar Infantis and of the receptor strains Escherichia coli K12 F^-, Lac⁺ and Nal^r (originally from the Biophysics Institute, Federal University of Rio de Janeiro) were incubated at 1:10 inoculum proportion at 37 °C for 18 hr. Transconjugants were plated onto Agar McConkey, with the antimicrobial drugs added individually at the following concentrations: ampicillin, 10 mcg/mL – gentamicin – 10mcg/mL; ceftriaxone – 16 mcg/mL. All plates contained nalidixic acid at the concentration of 30 mcg/mL.

The growth of lactose-positive colonies (transconjugants) indicated the transfer of resistance markers. This result was confirmed by an antimicrobial susceptibility test and plasmid profile analysis of the transconjugants, considering the original pattern of the corresponding donor colony.

Epidemiological analysis of patients

For each HI case an epidemiological report was done. It contained patients' data. These data were saved in an EXCEL 5.0 program (Microsoft) and analyzed later in Epi-Info Version 6.04b - January-1997.

RESULTS

According to the epidemiological report, several parameters were analysed. General features of 166 patients studied can be seen in Table 1. The average age was 33.4, and average number of days of hospitalization before the HI was 25.4 days. 72.9% of the patients used prophylactic or therapeutic antimicrobials before presenting a positive culture. In the case of polimicrobial infections, an average of 2.9 strains per anatomic site was detected. The risk conditions which were mostly related to the HI process were surgery (19.3%), HIV infection (18.1%), lung disease (16.9%) and prematurity (12.0%).

A total of 28 different types of antimicrobials (Table 2) were used prior to the positive culture, with a predominance of amikacin (11.9%), ampicillin (11.6%), gentamicin (11.0%) and ceftriaxone (10.0%).

24 different species were identified (Table 3), with higher frequency of S. aureus (21.0%) followed by P. aeruginosa (18.5%); Coagulase-negative staphylococci (9.7%) and K. pneumoniae (8.8%). The 50 S. aureus strains were mainly distributed in Internal Medicine Wards (IMW) and General Surgery Wards (GSW). 17 (38.6%) strains of P. aeruginosa were from IMW and 9 (20.4%) from GSW. The other species were uniformely distributed by other wards. We detected an outbreak of Salmonella serovar Infantis in the Neonatology Intensive Care Unity.

The number of bacterial strains isolated according to the type of clinical material cultured is shown in Table 4. S. aureus was the predominat bacterium in surgical wound, blood, vascular catheter and cutaneus lesions. P. aeruginosa were most frequently isolated in surgical wound abdominal secretions and enteric tract, Coagulase- negative staphylococci (CoNS), K. pneumoniae and A. baumanni were frequently isolated from blood.

Considering the resistance to antimicrobial agents, 36% of the S. aureus were resistant to oxacillin. The resistance of Gram negatives to the antimicrobials is shown in Table 5. We observed a high percentage of resistance to aminoglycosides (gentamicin and amikacin) and 3^rd generation of cephalosporins (Ceftriaxone and Ceftazidime). There was a high percentage of P. aeruginosa (59.5%) resistant to carbenicillin and of A. baumanni resistant to ciprofloxacin, in spite of the latter being a controlled antimicrobial. Imipenem was the most active agent against K. pneumoniae, the others Enterobacteriaceae (E. coli, E. cloacae, P. vulgaris, M. morganii, P. rustigianii and S. marcescens) were more resistant.

An outbreak of Salmonella serovar Infantis, occurred between 05/April -20/April/1996 affected nine hospitalized children in the high risk nursery (Table 6). These were studied in more detail. All of them had severe disease: six had respiratory problems and three had low body weight. The antimicrobial resistance and plasmidial DNA profiles of all the strains were identical, with only one plasmid (98 Mda). In the determination of the presence of R factors, all of the strains transfered the resistance plasmid to the standard strain recipient E. coli K12.

DISCUSSION

The HUGG provides assistance to adults, children and neonatology. It is, also, a reference hospital for some diseases such as: acquired immunodeficiency syndrome, diabetes and cancer. In this way, a large part of the patients, due to their underlying diseases are more especially susceptible to acquired hospital infections.

This bacteriological study, complemented with information about patients in hospital, had some aspects. The S. aureus (21%) was the predominant pathogen (Tables 3, and 4). SADER et al. (1999), in a multicentric study, related similar result in blood and surgical wounds infections.

In Table 1, it is interesting to point out the average number of days of hospitalization (25.4 days) before the diagnosis of HI, a long time of hospitalization, as well as, the long time of previous use of antimicrobials (15.9 days). These factors permit the emergence of multirresistant bacteria. MANRIQUE & GALVÃO (1996), reported that the use of prophylactic or therapeutic antimicrobials, may result in the spread of multirresistant strains in the hospital. Another aspect is that risk conditions or underlying diseases may have favored the occurrence of HI in some hospital areas, mainly in surgery wards and intensive care units. The Table 3 shows that wards SW, IMW, NICU and PICU sum up 36% of the places studied, according to SARTOR et al. (1995).

Polimicrobial infection (22.3% of the cases) represented a challenge in establishing proper treatment and emphasized the importance of obtain cultures for every bacterial infection in hospitalized patients before starting an empiric treatment. DUNCAN et al., (1994), mention that some infections demand empiric antimicrobial therapy; however, this type of therapy should be guided by previous study in vitro susceptibility of the most frequent the HI bacterial strains of higher frequency in the hospitals.

Regarding the frequency of the microorganisms, S. aureus and P. aeruginosa sum up 39.5% of the total isolates; Coagulase- negative staphylococci (CoNS), K. pneumoniae, E. coli and A. baumannii sum up 32.4%. The other 28.2% were represented by other 18 bacterial species. These microorganisms have been described in the literature as the main causes of HI in hospitals that provide tertiary assistance to HIV patients and high risk newborns (AYLIFFE, 1997; JARVIS, 1987; BERGOGNE-BÉRÉZIN, 1995; SARTOR et al., 1995; MONNET et al., 1997; STROUD et al., 1997; SADER et al., 1999). It is also important to point out that the distribution of microorganisms mostly involved in HI is directly related to the origin (wards) of the patients and type of clinical material. The knowledge of the hospital indigenous flora isolated from each anatomical sites allows a better choice of empirical treatment.

The level of multirresistance of the species of the KES group (Klebsiella sp., Enterobacter sp. and Serratia sp.) is worrying, since these microorganisms are rather frequent, distributed in several wards of the hospital. The multirresistance in this group is related to the resistance to ampicillin, cephalosporin (except cefoxitin), gentamicin and amikacin, with combinations of resistance to chloramphenicol, tetracycline and trimethoprim-sulfametoxazole. According to FRENCH et al. (1996), these strains of Klebsiella sp. do not respond to an empiric treatment with gentamicin or amikacin. LIVRELLI et al. (1996), emphasized that these bacterial strains are pathogens which cause infections, mainly on immuno-defficient patients.

36% of Methicillin-resistant S. aureus (MRSA) were found. This result matches the results of other places in Brazil and other countries (LEVY et al., 1991; PANLILIO et al., 1992; VOSS et al., 1994; DURMAZ et al., 1997; SADER et al., 1999).

Despite of the relatively recent use of imipenem and ciprofloxacin in hospital, we observed a considerable level of resistance to gram negatives. STRUELENS et al. (1993) verified that 40% of A. baumannii strains are resistant to the latter antimicrobial in Brussels.

The nosocomial outbreak caused by Salmonella serovar Infantis was the first one in the literature. It showed the potential virulence of a plasmid of high molecular weight, and the multirresistance of this serovar, similar to that of serovars Typhimurium and Agona in pathogenicity, virulence and potential to cause outbreaks of hospital infections.

Recent studies on salmonellosis show that Infantis and Agona serovars have been isolated in stools and blood of children in hospitals in Rio de Janeiro, with a significant prevalence of Typhimurium serovar (ASENSI, SOLARI & HOFER, 1994; ASENSI & HOFER, 1994). This S. Infantis outbreak probably resulted from the vulnerability of the neonates due to their severe primary processes which favored the infection of the bacterium. Among the risk factors (Table 6) were observed prematurity and respiratory problems at birth. Besides, all of the newborns were in a neonatal pathologic nursery, submitted to invasive procedures.

Another interesting aspect was the presence of the higher molecular weight plasmid (98Md), which was probably involved with codification of virulence genes in Salmonella sp. The presence of a 60Md plasmid of S. Typhimurium increases virulence in mouse models as compared with plasmid free or plasmid cured strains (JONES et al., 1982; JONES & OSBORNE, 1991). For some years, various publications have illustrated the role of plasmids as virulence related factors in various serovars of Salmonella. Chromosomal DNA is an important factor in expression of virulence, especially in the capacity of strains to survive and multiply in reticuloendothelial system cells. Nevertheless, the virulence of Salmonella strains is linked to a combination of chromosomal and plasmid DNA, the last one related to adhesion and invasion of HeLa cells and mouse infection (JONES et al., 1982; REXACH et al., 1994). In 1985, HELMUTH et al. characterized several virulence-associated plasmids in different Salmonella serovars.

The Committee for Hospital Infection Control used the results of this study to control new cases, optimize the use of antimicrobial in the hospital, implement its policies to reduce excess hospital stay and costs of medical care.

RESUMO

Análise epidemiológica de cepas bacterianas envolvidas em infecção hospitalar em um Hospital Universitário no Brasil

As infecções hospitalares representam um aumento na morbidade e mortalidade de pacientes internados, com significativo aumento no custo de internação hospitalar. Teve-se como objetivo fazer uma análise epidemiológica de casos de infecção hospitalar ocorridos num Hospital Universitário na cidade do Rio de Janeiro. Assim, foram analisadas 238 cepas isoladas a partir de 14 espécimens clínicos diferentes oriundos de 166 pacientes internados no período de 08 de 1995 a 07 de 1997. A idade média dos pacientes foi de 33,4 anos, 72,9% faziam uso de antimicrobiano antes de apresentar a cultura positiva, as patologias de risco mais comuns foram: Cirurgia (19,3%), HIV ou AIDS positivo (18,1%) e Patologia Pulmonar (16,9%). Foram identificadas 24 espécies bacterianas distintas, com predominância de S. aureus (21%) e P. aeruginosa (18,5%). Foram detectados 36% de MRSA (Methicilin Resistant S. aureus). Os Gram negativos apresentaram altos níveis de resistência para aminoglicosídeos e cefalosporinas. Foi detectado um surto de diarréia em berçário patológico, provocado pela Salmonella sorovar Infantis, com altos níveis de resistência para antimicrobianos e um plasmídio de alto peso molecular (98Mda), codificador do fator R.

Received: 15 September 1999

Accepted: 15 May 2000

Correspondence to: Dr. M.D. Asensi, Pav. Rocha Lima, 3º Andar, IOC/Fiocruz, Av. Brasil 4365, 21045-900 Manguinhos, Rio de Janeiro, Brasil.

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(1

) Laboratório de Enterobactérias, Departamento de Bacteriologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

(2

) Hospital Universitário Gaffrée e Guinle, Universidade do Rio de Janeiro, Brazil.

Publication Dates

• Publication in this collection
  31 Aug 2000
• Date of issue
  Aug 2000

History

• Accepted
  15 May 2000
• Received
  15 Sept 1999