Epidemiology of nosocomial colonization / infection caused by Acinetobacter spp . in patients of six surgical clinics in war and peacetime

Background/Aim. Acinetobacter spp. has emerged as nosocomial pathogen during the past few decades in hospitals all over the world, but it has increasingly been implicated as a serious nosocomial pathogen in military hospitals. The aim of this study was to analyse and compare the surveillance data on Acinetobacter nosocomial colonization/infection (NCI) collected during the wartime with the data collected in peacetime. Methods. We conducted a prospective study of incidence of Acinetobacter spp. colonization/infection. Also, the two nested case-control studies were conducted. The patients with nosocomial infection (cases) were compared with those with nosocomial colonization (controls) during the two different periods, wartime and peacetime. The patients with NCI by Acinetobacter spp. were identified by the case-based surveillance. The surveillance covered all the patients in 6 surgical clinics. Results. During the study periods a total of 166 patients had cultures that grew Acinetobacter spp. and the pooled rates of Acinetobacter spp. colonization and infection were significantly higher in wartime. When patients with NCI in wartime were compared with those with NCI in peacetime significant differences were observed. In the war year, the patients were more significantly males (p < 0.000). In a period of peace, most of the colonization/infections were reported from patients with certain chronic diseases (p = 0.020) and the survival of patients was more significant (p = 0.049). During the peacetime, proportions of Acinetobacter isolates resistent to ciprofloksacin, imipenem and meropenem were significantly higher (p < 0.001). Conclusion. This study provides additional important information about the risk factors of nosocomial Acinetobacter spp. infections in a large cohort of surgical patients. This is also the first study that directly examines epidemiological differences between NCI caused by Acinetobacter spp. during the war and peace period.


Introduction
Acinetobacter spp.has emerged as nosocomial pathogen during the past few decades in hospitals all over the world [1][2][3][4][5] , but it has increasingly been implicated as serious nosocomial pathogen in military medical facilities 6,7 .This Gram-negative coccobacillus is fairly stable and widely distributed both in nature and in hospital environment 8 .Acinetobacter may also colonize the skin of healthy humans 9 or patients without causing infection 10,11 .Its capacity to survive on most surfaces, animate and inanimate, suggests that hospital environment and colonized patients or healthcare personnel are the major reservoirs of this microorganisms.Multidrug resistance is common among these organisms and may complicate the treatment of serious infections.While resistance to antibiotics is not a new phenomenon for Acinetobacter, what is new is that in the last few years some strains have begun to develop resistance to the last effective group of active drugs called carbapenems, powerfull tools for management of nosocomial infections (NI) 12,13 .Types of infections due to Acinetobacter varied and included pneumonia, particularly ventilator-associated pneumonia (VAP) in intensive care units (ICU), bloodstream infections (BSI), surgical site infections (SSI), urinary tract infections (UTI), etc. 14 .Today, nosocomial colonization/infection (NCI) by Acinetobacter spp.are a real challenge to infectious diseases physicians, surgones and hospital epidemiologists, especially in wartime.
The aim of this study was to analyse and compare some surveillance Acinetobacter colonization/infection data collected during wartime with the data collected during peacetime.

Methods
The Military Medical Academy (MMA), Belgrade, Serbia, is a 1,200-bed tertiary center divided in 27 departments according to medical specialty.The surveillance of NCI by Acinetobacter spp.covered all patients in 6 surgical clinics of MMA: Plastic Surgery and Burns, Neurosurgery, Urology, Traumatology/Orthopedics, Cardio/Thoracic Surgery, General/Vascular Surgery.
We conducted a prospective analytic study.The patients with NCI by Acinetobacter spp.were identified by a casebased surveillance during the two different periods: wartime, June-September, 1999 (during NATO bombing of Serbia and 3 months after) and peacetime, June-September, 2000-2004.We gathered data on the following variables for patients with NCI: intrinsic factors, including gender, age, diabetes, neoplasm, chronic diseases (cardiac disease, pulmonary disease, hepatic disease, renal disease, malignacy, cerebrovascular accident) and survival; and factors related to health care: ICU admission, indwelling urinary catheter, central venous catheter, mechanical ventilation, nasogastric tube, previous surgery, transfusion of blood and blood products, previous antimicrobial treatment.
Clinical significance (colonization or infection) of Acinetobacter spp.isolation and the type of NCI in each surgical patient were assessed according to the Center for Disease Control (CDC) criteria 15 .
Clinical and microbiological data also recorded were: type of NCI, as well as antimicrobial sensitivity tests.The patients with isolation of Acinetobacter spp.recovered within 48 h of admission were excluded.Also, duplicate isolates from the same patient were excluded; these were defined as isolates of the Acinetobacter spp. with the same antimicrobial resistance pattern recovered from the same patient, regardless of the site of isolation.All the patients' characteristics with NCI in wartime were compared with those in peacetime.Also, the 2 nested case-control studies were conducted, with all the patients with NCI during the 2 different periods, wartime and peacetime.Those with NI (cases) were compared with those with NC (controls).
Microbiological testing for the hospital was performed at the Institite for Medical Microbiology in MMA.Identification of isolates was done using the routine methods 16 .Susceptibility testing was done according to the national standards in selection of antibiotics for antibiogram typing 17 .
Rates were estimated by dividing the number of new patients with NCI by the number of patient days or by the number of patient admissions in the study period.Statistical analysis for the data was done with SPSS software (SPSS, Chicago, version 10.00).The results were expressed as the mean + SD or as a proportion of the total number of patients.Testing for significant differences was conducted by the χ² test for categorical variables and by the Student's t-test for continuous variables in all studies.Factors were considered significant for a p-value of ≤ 0.05.All p-values are two tailed.Risk factors (RF) independently associated with infections in a period of peace and a period of war were identified by stepwise logistic regression analysis of variables selected by univariate analysis, with a limit for entering and removing variables at 0.05.

Results
During the study periods, 166 patients had cultures growing Acinetobacter spp.(96% were Acinetobacter baumannii [A.baumannii]), out of which 133 patients met NI criteria.Acinetobacter spp.pooled rates are shown in Table 1.The mean age of the study population was 44.33 years (range 10-86 years, median 41.50 years).The patient's characteristics and characteristics related to health care are shown in Table 2.There were no significant differences between the infected and colonized patients, or patients treated in wartime and peacetime, regarding the prior use of antibiotics (Table 3).Univariate analysis of potential RFs for infection by Acinetobacter spp. in the wartime in MMA, showed that the patients with NI were more significantly hospitalized in the ICU, and significantly more of then had a urinary catheter and transfusion of blood and blood products than patients with NC (Table 4).But, transfusion of blood and blood    sites were equally distributed in both groups of patients (wartime and peacetime) (p = 0.161) (Table 7).
Antibiotic susceptibilities of all isolates in the war and peacetime were presented in Table 8.There were no significant differences in the susceptibility frequencies for amikacin, gentamicin, ceftazidim and ampicilin/sulbactam in two study periods.During peacetime, proportions of Acinetobacter isolates resistent to ciprofloksacin, imipenem and meropenem were significantly higher (p < 0.001).

Discussion
In the available literature we found a number of studies regarding epidemiology, clinical features, resistance to antimicrobial agents of Acinetobacter spp infection/colonization.This study provides further important information about the RF of nosocomial Acinetobacter spp.infections in a large cohort of patients admitted to 6 surgical clinics of military tertiary care center in Serbia.Also this is the first study that directly examines epidemiological differences between NCI caused by Acinetobacter spp.during war and peace period.
Worldwide, during peacetime conditions, NI constitutes one of the greatest challenges of modern medicine.War conditions imply numerous and different injuries.Generally, war wounds are grossly contaminated, and high velocity missiles can cause massive destruction of bones and soft tissue.Extensive burn injures may be isolated or associated with severe trauma.In addition to trauma, and as direct consequence of war-related circumstances, NIs become one of the greatest challenges of military medicine, too.Some new studies emphasized that war wounds, immediately after injury during the current Iraq conflict, showed the presence of a range of less pathogenic, Gram-positive, skin-commensal bacteria.At the same time Gram-negative bacteria were rarely found, and none was multidrug resistant.So, the use of broad-spectrum antibiotics is unnecessary, but effective infection-control practice to prevent colonization with possible subsequent infections is necessary in early wound management 18 .
Our study showed that the pooled rate of Acinetobacter NCI was 0.9 cases per 100 admissions or 0.7 cases per 1,000 patient days in peacetime.During the period of war this pooled rate was higher, 3.0 cases per 100 admissions or 2.1 cases per 1,000 patient-days.A large CDC study on Acinetobacter epidemiology conducted from 1987 to 1996 reported the average rates of infections of 0.72 cases per 1,000 patient days in ICUs, with persistant seasonal variation in the rate of infections, which increase in late summer months for all major infection sites 19 .During Novemeber 2000, in 25 Spanish hospitals, pooled rate in general hospitals was 0.39 cases per 1,000 patient days with highest rate in ICUs 3 .In our study, the highest Acinetobacter NCI rates were in Plastic Surgery and Burns Clinic, both in peace and war period (1.3 cases vs 5.2 per 100 admissions or 1.3 vs 3.9 cases per 1,000 patient days, respectively).Today, it is well-known that patients with burn injures reperesent a highly susceptible population.The outbreaks of infection caused by A. baumannii infection have been reported in burn units.During the outbreak due to multiresistant A. baumannii in Ross Tilley Burn Centre, rate of colonization/infection was 12.6 per 100 admissions 20 .
Our study also showed significant differences between patients with NCI in wartime and those with NCI in peacetime.In the war year, patients were more significantly males.Similar situation regarding gender was described during the period January 1, 2002 to August 31, 2004, when military health officials reported BSI in 102 patients at 2 military medical facilities, Landstuhl Regional Medical Center (LRMC) in Germany and Walter Reed Army Medical Center (WRAMC), USA, in which service members injured in the  Iraq/Kuwait region and in Afghanistan were treated.Of the 33 patients with A. baumannii BSI at LRMC and of the 45 patients with A. baumannii BSI at WRAMC, 32 (97%) and 39 (87%) were males, respectively 7 .In the study of Lee et al. 21, conducted in patients admitted during 2000 at the Asan Medical Center in Seoul, Korea, male gender was identified as independent RF for nosocomial occurrence of imipenemsusceptible, but not for nosocomial occurrence of imipenemresistant A. baumannii.In the wartime the 100% of our isolates were susceptible to imipenem.Furthermore, as patients in hospitals all over the world 22 , our patients with some chronic diseases were at the greater risk for NCI in the period of peacetime.
Sunshine et al. 23 performed retrospective, matched cohort investigation at two Baltimore hospitals, to examine outcomes of patients with multidrug (MDR) Acinetobacter infection compared with patients with susceptible Acinetobacter infections and patients without Acinetobacter infections.They showed that 92% of their MDR isolates were not susceptible to carbapenems.Also mortality hospital rate for patients with MDR Acinetobacter infections (26%) were higher than for susceptible references (18%) and uninfected references (11%), but only the difference between MDR Acinetobacter-infected patients and uninfected patients was statistically significant.Some study conducted in intubated patients, showed that pneumonia caused by Acinetobacter spp. is not significantly associated with attributable mortality rate 24 .Proportions of Acinetobacter isolates resistent to imipenem and meropenem, were significantly increased during the peacetime, in our study, but the patients survival was significantly higher in this period than in wartime.
Acinetobacter spp. was the most frequently recovered Gram-negative isolate from war wounds and the second most frequent bacterium causing BSI in US Marines with extremity wounds during the Vietnam War 25 .Injured soldiers from Iraq and Afghanistan have also brought an epidemic of MDR A. baumannii infection to military hospitals 7 .First, it was not known how this has occurred and the reservoir for infection was unclear, but recent investigation by the military medical and research community suggests that these are NI 7,18,26,27 .During wartime, this bacteria was prevalent isolate in our hospital, too 6 .
Univariate analysis of potential RFs for infection by Acinetobacter spp. in wartime in MMA, showed that patients with NI were more significantly hospitalized in the ICU and significantly more often had a urinary catheter and transfusion of blood and blood products than patients with NC.But, transfusion of blood and blood products was the only one independent RF for NI of patients in wartime.Also, a study which analyzed data on 5,366 patients over a 2-year period, showed the receipt of blood products as an independent risk factor for infection in hospitalized trauma patients.There has been controversy as to wether such an association merely represents severity of underlying illness or wether receipt of blood products may induce immunosuppressive effects 28 .Some early works of Blumberg and Heal 29 , showed that infections associated with transfusions were the major factor in altering host immune defenses 29 .A Canadian national universal leukoreduc-tion program was assocated with unadjusted in-hospital mortality rates, decrease of fever episode, and antibiotic use, but number of serious NI did not decrease 30 .
Potential RFs for infection in our patients caused by Acinetobacter spp. in peacetime were similar to those in wartime.Our patients with Acinetobacter NI were more significantly hospitalized in the ICU, and significantly more often had a urinary catheter and received transfusion of blood and blood products than patients with NC.Multivariate logistic regression analysis identified hospitalization in the ICU as the only one independent RF for NI of patient in peacetime.
Similar multiple factors have also been found in previous studies conducted during peace period all over the world 20,22,31,32 .
In the past decades in the USA hospitals, Gram-negative bacilli have been the most frequent pathogens associated with pneumonia and UTIs.However, Gram-positive bacterial pathogens were most frequently associated with BSIs and SSIs.The proportion of ICU pneumonia episodes associated with Acinetobacter spp.significantly increased from 4% in 1986 to 7% in 2003 33 .SSI, UTI and burns associated with Acinetobacter spp.were more frequent among the patients treated in MMA in war time, but pneumonia, BSI, and decubitus ulcer associated with Acinetobacter spp.were more frequent among the patients treated in peace time (p  0.025).
Acinetobacter spp.isoaltes have inherent and/or easily acquired mechanisms of resistance aganist many of the available antimicrobial agents, which make this pathogen one of the most significant microbial challenges of the current era.Differences in antibiotic susceptibility have been observed among countries, probably as a result of different patterns of antimicrobial usage and environmental factors, especially infection control practice 14,27 .Although in our hospital, broad spectrum antibiotics are extensively used there were no significant differeces between infected and colonized patients, or patients treated in wartime and peacetime, regarding prior use of antibiotics.Also, there were no significant differences in frequencies for amikacin, gentamicin, ceftazidim and ampicilin/sulbactam susceptibility in the two study periods.During peacetime, proportions of Acinetobacter isolates resistant to ciprofloksacin, imipenem and meropenem were significantly higher.Most of Acinetobacter isolates in France, which were originally susceptible to fluoroquinolones, became resistent within 5 years of introduction of these antibiotics 14 .In a report of a citywide clonal outbreak in New York City, 53% of A. baumannii were resistant to meropenem/imipenem and 12% were resistant to all standard antibiotics 13 .
Acinetobacter spp.has been nicknamed the "Gramnegative MRSA", but the same measures cannot be used to control these two pathogens.Therapeutic approaches and control measures should be customized according to epidemiologic pattern of resistant organisms typical for each institution.So, a decision on how to prevent transmission and how to treat patients at risk is the most important step in struggle against this nosocomial pathogen 34 .
The most important limitation of our systematic review is that we were unable to type isolates using molecular meth- ods.The use of molecular typing methods in epidemiologic studies is crucial for investigation sucess.

Conclusion
When characteristics of surgical patients and characteristics related to the health care of surgical patients colonized or infected with Acinetobacter spp. in wartime and peacetime are compared, significant epidemiologic differences are observed.
Our results suggest that pooled rates of Acinetobacter spp.colonization and infection, especially NI as SSI, UTI's and NI of burns, were significantly higher during wartime.The only independent RF for NI in patients was blood and blood products transfusion during wartime and ICU admission during peacetime.No significant differences regarding the prior use of antibiotics were observed between infected and colonized patients or patients treated during war and peacetime, but proportions of Acinetobacter isolates resistant to ciprofloxacin, imipenem and meropenem were significantly higher during peacetime.

Table 2 Characteristics of the patients and characteristics related to helath care of patients colonized or infected with Acinetobacter spp. in wartime (1999) and peacetime (2000-2004) in the Military Medical Academy, Belgrade
*Clinics for: PlastigcSurgery and Burns, Neurosurgery, Urology, Traumatology/Orthopedics, Cardio/Thoracal Surgery, General/Vascular Surgery

Table 3 Previous use of antibiotics
SSI, UTI and burns were more frequent among the patients in wartime, but pneumonia, BSI, and decubitus ulcer were more frequent among the patients in peactime (p  0.025) (Table6).Clinical isolates from different culture *Clinics for: PlastigcSurgery and Burns, Neurosurgery, Urology, Traumatology/Orthopedics, Cardio/Thoracal Surgery, General/Vascular Surgery OR -odds ratio; CI -confidence interval Šuljagić V, et al.Vojnosanit Pregl 2011; 68(8): 661-668.products was the only one independent RF for the NI of the patients in a wartime [Odds ratio (OR): 6.2, 95% confidence interval (CI): 1.6-24.1;p=0.008].Univariate analysis of potential RFs for infection of our patients by Acinetobacter spp. in peacetime were similar to those in wartime (Table5).Also, multivariate logistic regression analysis identified hospitaliza-tion in the ICU as the only one independent RF for the NI of patient in peacetime (OR: 6.4, 95% CI: 1.4-30.4;p = 0.018).

Table 4 The results of univariate analysis of potential risk factors for infection by Acinetobacter spp. in the Military Medical Academy, Belgrade in wartime (1999)
*Clinics

Table 5 The results of univariate analysis of potential risk factors for nosocomial infection by Acinetobacter spp. in peacetime (2000-2004) in the Military Medical Academy, Belgrade
*Clinics

Table 7 Culture sites of clinical isolates of Acinetobacter spp. in wartime (1999) and peacetime (2000-2004) in the Military Medical Academy, Belgrade
*Respiratory cultures include bronchoalveolar lavage, sputum and tracheal aspirat