Endemic carbapenem-nonsusceptible Acinetobacter baumannii-calcoaceticus complex in intensive care units of the national referral hospital in Jakarta, Indonesia

A prospective observational study was performed at the Dr. Cipto Mangunkusumo General Hospital, a 1000-bed teaching hospital in Jakarta, Indonesia, from April–October 2013 and from April–August 2014. We conducted this study in two ICUs: the 12-bedded adult ICU and the 8-bedded Emergency Room (ER)-ICU with an average of 1010 and 415 admissions per year, respectively. The adult ICU is an open ward with mechanical ventilation facilities, admitting patients with mixed medical and surgical indications, and one designated nurse per patient during first shifts (7 am-3 pm) and a 1:1.5 nurse/patient ratio during other shifts. The ER-ICU has the same design, and the nurse-to-patient ratio in the first shifts is 1:1 and during the other shifts 1:2. The populations served by these two ICUs were identical, and there was also no difference in the service provided.

The study was performed in the framework of a larger study that focused on carbapenem-nonsusceptible Klebsiella pneumoniae, Pseudomonas aeruginosa, and A. baumannii-calcoaceticus complex.

All adult patients (≥18 years old) admitted to one of the two ICUs and hospitalized for more than 48 h were eligible for enrollment in this study. The first screening cultures were taken on the day of admission, and if a patient was discharged before 48 h, he or she was excluded. Informed consent was obtained from the patient or their relatives as applicable. Demographic and clinical characteristics such as age, gender, medical or surgical indication, underlying diseases, hospitalization history, and previous use of antibiotics were recorded on admission.

Systemic inflammatory response syndrome (SIRS) criteria on admission were used as a screening tool to assess (severity of) septic illness. The SIRS criteria were calculated and included in the study, as this was practice at the time of the study [6].

The quick Sequential Organ Failure Assessment (qSOFA) score is a new bedside prompt that may identify patients with suspected infection and helps to determine sepsis in all healthcare environments. The qSOFA score assigns one point for each of the following conditions: systolic blood pressure ≤ 100 mmHg, respiratory rate ≥ 22 breaths per minute, and altered mentation (Glasgow coma scale <15). A qSOFA score ≥ 2 at the onset of infection is associated with a greater risk of death and prolonged ICU stay [6].

The primary outcome measure was acquisition of a carbapenem-nonsusceptible A. baumannii-calcoaceticus complex. Acquisition is defined as a screening culture or clinical culture with first detection of A. baumannii-calcoaceticus complex, with reduced susceptibility to a carbapenem, that was not present within the first 48 h of admission. Secondary outcome measures were length of stay in the ICU, and mortality during ICU stay.

Environmental samples (Additional file 1: Table S1), were taken twice (in October 2013 and December 2014), simultaneously in both ICUs. Screening of healthcare workers (HCWs) was performed once. HCWs were defined as all personnel including doctors, nurses and other people (cleaning staff, administration staff, porters, nutritionist) working in one of the two ICUs during the study period.

From patients enrolled, screening cultures were obtained from throat and rectum or stools by experienced ICU nurses who had been trained for the task of taking the samples, on the day of admission, at the time of discharge from the ICU, and weekly if the patient was admitted for seven days or more. Sampling was performed using sterile cotton-tipped swabs, and swabs were transported to the laboratory in Amies transport medium (Oxoid, Basingstoke, UK). The swabs in medium were transported in clean, closed boxes at ambient temperature to the laboratory on the same day. All swabs were processed in the laboratory within 24 h.

Clinical samples were collected on indication from patients under aseptic precautions from the lower respiratory tract, blood, urine, tissue, or wound.

Environmental samples were taken from various sites, including washbasins, bed rails, bedside cabinet tables, ventilators, and monitor screens (Additional file 1: Table S1), with sterile cotton-tipped swabs and placed in Amies transport medium.

All HCWs working in one of the ICUs were sampled once over the course of one month (September 2013) with sterile cotton-tipped swabs, which were transported to the laboratory in Amies transport medium.

Statistical analyses were done using SPSS Version 24.0 (SPSS, Chicago, IL, USA). Patients admitted to adult ICU were compared to ER-ICU using Chi square or Fisher’s Exact and Mann-Whitney as appropriate. One-way ANOVA was used to compare patient characteristics according to their A. baumannii-calcoaceticus complex status. Univariate and multivariate analyses were performed to establish risk factors associated with mortality using a multivariate logistic regression model with backward selection and inclusion of variables with a p-value <0.1 in the univariate analysis. Cox proportional regression was used to analyse risk factors for length of stay. Kaplan-Meier method was performed to construct survival curves. P-values of less than 0.01 were considered significant [14].

During the 11-month study period, 1211 patients were hospitalized in the ICUs (Adult ICU: 863, ER-ICU: 348). Additional file 1: Table S2 shows baseline characteristics of patients in each ICU. Of the 412 included patients, 188 were admitted to the adult ICU and 224 to the ER-ICU. There were no significant differences in characteristics between patients in both ICUs, except that in the adult ICU most of the patients had been referred from another ward in the same hospital (Additional file 1: Table S2). Therefore, we analyzed the data from the ICUs both separately and pooled.

Overall, 158/412 (38.3%) patients had a positive culture with carbapenem-nonsusceptible A. baumannii-calcoaceticus complex, the remaining 254 patients were free from carbapenem-nonsusceptible A. baumannii-calcoaceticus complex on admission and remained so during their ICU stay. Sixty-nine patients (69/412; 16.7%) already carried carbapenem-nonsusceptible A. baumannii-calcoaceticus complex as revealed by screening cultures taken on the day of ICU admission, 89/343 (25.9%) patients who were initially culture-negative acquired carbapenem-nonsusceptible A. baumannii-calcoaceticus complex during their ICU stay (Additional file 2: Figure S1). Of the total of 158 patients with positive cultures, the positive cultures were obtained from screening specimens only in 80 patients, from clinical specimens only in 34 patients and from both screening and clinical samples in 44 patients. Interestingly, of the patients that were positive on ICU admission, 17 (24.6%) were admitted directly from the emergency unit. Six patients had one or more blood cultures with carbapenem-nonsusceptible A. baumannii-calcoaceticus complex, and three of them died on the ICU. The dynamics of acquisition of carbapenem-nonsusceptible A. baumannii-calcoaceticus complex in the ICU is shown in Fig. 1, 60% of patients that became positive for carbapenem-nonsusceptible A. baumannii-calcoaceticus complex during their ICU stay did so in the first week of ICU stay. There were no differences in the dynamics of carbapenem-nonsusceptible A. baumannii-calcoaceticus complex acquisition between the two ICUs (median acquisition day in adult ICU: 7, in ER-ICU: 6). The acquisition rate for carbapenem-nonsusceptible A. baumanni-calcoaceticus complex was 43 per 1000 patient-days at risk overall, with an average of 43 per 1000 patient-days in the adult ICU and 43 per 1000 patient-days in the ER-ICU.

Patient outcomes were clearly associated with carbapenem-nonsusceptible A. baumanni-calcoaceticus complex status of patients. Patients who acquired carbapenem-nonsusceptible A. baumannii-calcoaceticus complex during their ICU stay had a significantly longer length of stay (median [interquartile range (IQR)]: 11 [5‐18], adjusted hazard ratio [aHR]: 2.56 [99% confidence interval (CI): 1.76–3.70], p < 0.001, Additional file 1: Table S4, particularly the group of patients that became positive before the day of their discharge (median [IQR] 13 [8‐23] days, p < 0.001, Fig. 2) compared to the other groups of patients, of which ≥80% were discharged from the ICU within ten days. Interestingly, these latter groups not only included the patients that were always free from carbapenem-nonsusceptible A. baumannii-calcoaceticus complex, but also included patients that already carried carbapenem-nonsusceptible A. baumannii-calcoaceticus complex at the time of admission to the ICU, and patients that remained free of carbapenem-nonsusceptible A. baumannii-calcoaceticus complex until they were found to be positive by screening on the day of their discharge from the ICU (Fig. 2).

Acquisition of carbapenem-nonsusceptible A. baumannii-calcoaceticus complex was not associated with mortality, 23.2% of patients that remained free of carbapenem-nonsusceptible A. baumannii-calcoaceticus complex died versus 42.7% of patients that acquired carbapenem-nonsusceptible A. baumannii-calcoaceticus complex during their ICU stay (Fig. 3, p = 0.066; Additional file 1: Table S3, multivariate analysis: adjusted Odds Ratio (aOR):1.59 [99%CI: 0.74–3.40]). Importantly, the admission SIRS and qSOFA scores of patients with or without A. baumannii-calcoaceticus complex acquisition did not differ (Table 1), indicating that the difference in the risk of dying was not present at the time of ICU admission but emerged later during their ICU stay (SIRS: crude Odds Ratio (cOR):1.69 [99%CI:0.55–5.22], p = 0.230; qSOFA:cOR: 1.45[99%CI:0.68–3.08], p = 0.211, Additional file 1: Table S3).

Patients that were free of carbapenem-nonsusceptible A. baumannii-calcoaceticus complex during their entire ICU stay were less likely to have had prior exposure to antibiotics, especially carbapenems (p < 0.01), they were more likely to have had a surgical indication for their admission to the ICU, and less likely to have had cerebrovascular disease (Table 1). Patients that acquired carbapenem-nonsusceptible A. baumannii-calcoaceticus complex during ICU stay had undergone a procedure (mechanical ventilation), had a medical device (central venous catheter or urine catheter) or had received carbapenem therapy more often than the other groups in the univariate analysis (p < 0.01) (Table 1). In a multivariate comparison of patients who acquired carbapenem-nonsusceptible A. baumannii-calcoaceticus complex to patients that were always negative, only carbapenem therapy during ICU admission could be identified as a risk factor (aOR: 3.37 [99%CI: 1.68–6.77], p < 0.01).

In total, we collected 311 carbapenem-nonsusceptible isolates from 158 patients, six carbapenem-nonsusceptible A. baumannii-calcoaceticus complex isolates cultured from the environment (table, bed rails, sinks, and tapwater), and a single isolate from a healthcare worker (throat) that was carbapenem-nonsusceptible as well (Additional file 1: Table S5).

The bla_OXA-23-like gene was demonstrated in 292/318 (91.8%) isolates including isolates from patients, the environment and from the healthcare worker. The bla_OXA-24-like gene was detected in a single isolate. Coexistence of OXA-23 with other oxacillinases and carbapenemases was found: OXA-23/OXA-58 (1 isolate), and OXA-23/NDM-1 (4 isolates). The bla_OXA-23-like gene was always demonstrated in combination with the ISAba1 insertion element upstream to the OXA-23 beta-lactamase. The intrinsic A. baumannii-calcoaceticus complex gene bla_OXA-51-like was demonstrated in all isolates. In the subset of isolates that were subjected to WGS (n = 14), the bla_OXA-51-like gene involved was bla_OXA-66 in 13 isolates and bla_OXA-68 in one isolate (Table 2).

Raman spectroscopy analysis performed for all of the isolates, revealed the presence of multiple types within the collection of A. baumannii-calcoaceticus complex. In total, 51 Raman types were identified. Interestingly, the majority of strains belonged to one of five major clusters (Additional file 3: Figure S2). The largest cluster (designated CIPTO-31) consisted of 111 isolates obtained from 69 patients (screening and clinical specimens) and four isolates from the environment. The sources of the five major clusters are specified in Additional file 1: Table S6. Strains belonging to the dominant cluster CIPTO-31 were present in both ICUs throughout the study period, whereas other clones seemed to wax and wane over time (Fig. 4). Patients were colonized with carbapenem-nonsusceptible A. baumannii-calcoaceticus complex irrespective of the location of their bed in these ICUs indicating that spreading of carbapenem-nonsusceptible A. baumannii-calcoaceticus complex in the ICUs was not restricted to only a part of the ICU (Additional file 4: Figure S3).

MLST, performed for a subset of 14 isolates, revealed the presence of multiple sequence types (STs), which corresponded closely to the Raman spectroscopy clustering (Table 2). Four previously identified STs (ST195, ST208, ST218, and ST642) as well as several new STs, and a new allele for the gpi gene were found in this study (Table 2).