Mahmoud Sadeghi-Haddad-Zavareh, Hadi Ahmadi Jouybari, Mostafa Javanian, Mehran Shokri, Masomeh Bayani, Mohammad Reza Hasanjani Roushan, Arefeh Babazadeh, Soheil Ebrahimpour, Parviz Amri Maleh

1Infectious Diseases and Tropical Medicine Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, I.R. Iran

2Department of Anesthesiology, School of Medicine, Babol University of Medical Sciences, Babol, I.R. Iran

1. Introduction

Ventilator-associated pneumonia (VAP) is one of the most common infectious complications and the leading cause of death in intensive care units (ICUs)[1,2]. VAP based on time event is divided into two types: early VAP that occurs within 4 d and late VAP which happens after the 5thday of hospitalization[3]. Risk factors of VAP include oropharyngeal colonization, trauma, surgery,imunosuppression, old age, urgent intubation, prolonged admission in ICU, sedative drugs steroids usage and previous hospitalization.Prevalent etiological agents in generating VAP in several studies consist ofStaphylococcus aureus (S. aureus),Pseudomonas. aeruginosa(P. aeruginosa),Acinetobacter baumannii (A. baumannii)[4,5].Generally, etiological agents of VAP are different based on hospital or geographical position and their antibiotic resistance rates is also different among various areas[6]. The results of the different studies show that resistance rates of bacteria are increasing[7]. Increasing resistance to antibiotics raises the mortality rate, admission duration and expenses, in patients who suffer VAP in ICU. The mortality rate in VAP was reported 20%– 76% in different studies[8,9].

In various studies ofP. aeruginosaandA. baumanniiin VAP the mortality of was 65% and 87% and for MRSA to was 84%[10].

In some study, using antibiotics of choice for treating VAP based on antibiotic resistance pattern in the same hospitals could decrease usage of inappropriate antibiotics and increase treatment success[11]. Considering that a study based on Bronchoscopy sampling method and bronchoalveolar lavage (BAL) and quantitative culture performance and investigating microorganisms resistance with microdilution method and determining minimal inhibitory concentration (MIC) of antibiotic, has not been carried out so far in ICU of Ayatollah Rouhani Hospital of Babol (northern Iran), this study was done with the purpose of determining microorganisms involved in creating pneumonia from ventilation and their antibiotic resistance evaluation noticing method above.

2. Materials and methods

This cross sectional study was conducted on 50 patients suffering ventilator associated pneumonia in ICU of Ayatollah Rouhani Hospital of Babol during the 2014–2015.

In this study with daily visit of hospitalized patients in Ayatollah Rouhani ICU, considering clinical criteria based on Clinical Pulmonary Infection Score (CPIS), patients who at least acquired six points based on CPIS and from the clinical signs, diagnosis of pnenumonia was possible for them, were under sampling method BAL and bronchoscopy[12]. The samples were cultured in blood agar and MacConkey agar, and culture media after 24–48 h incubation at(35±2) °C were evaluated. Samples were cultured under quantitative method and if growth of more than 104CFU/mL bacteria were detected presumped as VAP etiological agents, in next step for determining sensitivity of microorganisms, Broth microdilution method was used. Ninety six part microplate which was applied in this method, has 12 columns that hole of the 11thcolumn as negative control and the 12thcolumn as positive control were used. In hole of the first group and the first hole of negative control, 200 mL of brain heart infusion (BHI) culture area of broth were poured and then in the rest of holes, 100 mL of BIH broth area were added. In the next step, antibiotic were added to the entire first column hole and the first hole of negative column and then based on standard method, suitable dilution of antibiotic in holes were prepared and after that diluted bacteria suspension to 0.1 and 5 mL of bacteria was added to holes except the negative control hole. Eventually,the final volume of all holes was 100 mL. The negative control was without bacteria and positive control was without antibiotic. Then micro plate were incubated in the temperature of 37 ℃ for 24 h.After 24 h micro plate were investigated under the light of the lamp and the last hole which turbidity wasn’t seen in it, was considered as MIC and by comparison with the table CLSI 20/3, resistance,semi-sensitivity or sensitivity of the bacteria relative to antibiotic was reported[13]. In this study, the investigated antibiotics for grampositive bacteria included nafcillin, cloxacillin, co-trimoxazole,cefazolin, vancomycin and the investigated antibiotics for gramnegative bacteria included ciprofloxacin, ceftazidime, piperacillin/tazobactam, gentamycin, amikacin, cefepime, clavulanate/ticarcillin,meropenem and imipenem.

All of the applied antibiotics in this recent study were produced and made by German company Merk and information yield from.All data were analyzed by SPSS software 16.

3. Results

Of the 50 patients who suffer VAP in our study, 33 (66%) are male and 17 (34%) were female. The mean age of the patients was 67.43 year old. Among the patients 52% previously admitted in hospital and 60% had a history of antibiotic use in past 3 months(Table 1).the most common cause of admission of patients was neurologic disease (36%) and then respiratory disease. 12% of patients admitted with sepsis. And16%of patients was on stroid therapy. Demographic data of the patients are shown in Table 1.The most common microorganisms in our study wereA. baumannii(70%),P. aeruginosa(12%),S. aureus(8%) andKlebsiella pneumonia(K.pneumonia)(6%). Meanwhile, from these 50 investigated samples, two samples (4%) did not grow in culture media.

3.1. Evaluation of antibiotics resistance based on microdilution method in Acinetobacterbaumannii

Five antibiotics did not have any effect on 35 samples ofA.baumanniiin our study including that ciprofloxacin, ceftazidime,amikacin, clavulanate/ticarcillin and cefepime. Meropenem,imipenem and piperacillin/tazobactam had 97.1% resistance respectively, and gentamycine had 94.3% resistance (Table 1).

Table 1Resistance pattern of antibiotics in A. baumannii [n, (%)].

3.2. Evaluation of antibiotic resistance based on micro dilution method in P. aeruginosa

In our study, the most resistance was related to ceftazidime,clavulanate/ticarcillin, cefepime, (each with 66.7% resistance) and the least resistance was related to imipenem (16.7%) and gentamycin(16.7%) (Table 2).

Table 2Resistance pattern of antibiotics in P. aeruginosa [n, (%)].

3.3. Evaluation of antibiotic resistance based on microdilution method in K. pneumonia

Among the three samples ofK. pneumoniain our study, the most resistance was related to ceftazidime, cefepime and clavulanate/ticarcillin eaach with 100% of resistance. Gentamycin, meropenem,imipenem, piperacillin/tazobactam and ciprofloxacin each with 33.3% had the least resistance (Table 3).

Table 3Resistance pattern of antibiotics in K. pneumonia[n, (%)].

3.4. Evaluation of antibiotic resistance based on microdilution method in S. aureus

Among the four samples ofS. aureusin our study, resistance to cloxacillin and nafcillin was 75%. 25% of cases were sensitive to cotrimoxazole and no complete resistance to vancomycin was reported(Table 4).

Table 4Resistance pattern of antibiotics in S. aureus [n, (%)].

4. Discussion

Considering the extension of antibiotic resistance, early diagnosis of VAP and identification of the type of microorganisms and antibiotics resistance pattern, can modify the method of antibiotic prescription and as result decrease medication resistance. In our study the most common microorganisms of causing VAP wereA.baumannii,P. aeruginosaandS. aureusthat these results are similar to other studies[14-16].

The resistance rate ofA. baumanniito carbapenemes like imipenem and meropenem in our study was 97.1% and in study done with Balkhyet al., in Saudi Arabia was 64.1% and in study of Salehifaret al., in Imam khomeili hospital of sari (Iran) was 100%[15,16]. In current study, the resistance rate ofP. aeruginosato carbapenem as meropenem was 33.3% and to imipenem 16.7%,while the resistance rate to carbapenem was reported 14.7% in study of Jamaatiet al, that was accomplished in Masih Daneshvari hospital of Tehran and it was reported 32.8% by Balkhyet al., in Saudi Arabia[16].

The resistance rate ofS. aureusin our study to nafcillin and cloxacillin was 75% and it was reported 80%, 41.1%, 65.4% and 66.7% bysome studies[16-18]. One of the features of this study in comparison with many other studies, like the study was carried outby Aziz Japoniet al., in shiraz in 2008–2009 and Balkhyet al.,study that was done in Saudi Arabia in 2004 to 2009 was related to sampling method that in our study bronchoscopy method and BAL were applied which were often more precise than ETA method[16,19].Also, in our study one of the characteristics was using CPIS and performing quantitative culture in order to positive consideration of BAL sample with colon of more than 104cfu/mL[10,20]. The value of quantitative culture was much more than qualitative culture for diagnosing and deciding to start the treatment of VAP.

The prevalence ofA. baumanniiin our study was 70% and it was reported by some studies 35.1%, 29% and 18% which shows that high prevalence ofA. baumanniias a producing organism of VAP at our hospital relative to the other studies and can be a serious warning in outbreaks of hospital acquired infections caused byA.baumannii[15, 17].

The clear role ofA. baumanniitypes among gram-negative microorganisms in hospital acquired infections like bacteremia,urinary tract infection, soft tissue infections and especially VAP and also high ability of these microorganisms in generating antibiotic resistance with various mechanisms, now a days is a major problem[21,22]. Various studies about antibiotic resistance ofA. baumanniiwas carried out which often they have reported high resistance of this microorganism[23,24].

In many countries, in case of severe infections ofA. baumannii.Use of carbapenems as a treatment choice is a rule but resistance toward them is also increasing[25-27].

The most common microorganisms involved in wereA.baumannii,P. aeruginosaandS. aureusthat among themA.baumanniiwas much more one and antibiotic resistance on all of the investigated antibiotics was about 100%. Noticing the results, high resistance of gram negative organisms to ceftazidime, cefepime and clavulanate/ticarcillin makes their use in empirical treatment of the VAP patients not appropriate.

坑道工程口部伪装是工程防护重要研究方向之一，一些工程在施工过程中破坏了原有地貌，伪装时需要轻质仿石器材来模拟口部附近的真山石，造成口部是一些普通石块的假象，从而形成有效欺骗。随着园林行业的发展、仿石技术的进步，在可见光波段模拟山石已非难事。然而军事侦察主要波段除了可见光外还有近红外、热红外、雷达等波段[1-3]，而真山石的光学、热红外和雷达波段特征具有独特性，园林仿石很容易被揭露。所以，能起到伪装作用的仿石器材需要一些新技术、新材料才能达到对抗多频谱侦察的需要。

Conflict of interest statement

The authors declare that there is no conflict of interest.

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