The role of the genetic elements bla oxa and IS Aba 1 in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex in carbapenem resistance in the hospital setting

Introduction: Members of the Acinetobacter genus are key pathogens that cause healthcare-associated infections, and they tend to spread and develop new antibiotic resistance mechanisms. Oxacillinases are primarily responsible for resistance to carbapenem antibiotics. Higher rates of carbapenem hydrolysis might be ascribed to insertion sequences, such as the ISAba1 sequence, near blaOXA genes. The present study examined the occurrence of the genetic elements blaOXA and ISAba1 and their relationship with susceptibility to carbapenems in clinical isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. Methods: Isolates identifi ed over 6 consecutive years in a general hospital in Joinville, Southern Brazil, were evaluated. The investigation of 5 families of genes encoding oxacillinases and the ISAba1 sequence location relative to blaOXA genes was conducted using polymerase chain reaction. Results: All isolates presented the blaOXA-51-like gene (n = 78), and 91% tested positive for the blaOXA-23-like gene (n = 71). The presence of ISAba1 was exclusively detected in isolates carrying the blaOXA-23-like gene. All isolates in which ISAba1 was found upstream of the blaOXA-23-like gene (n = 69) showed resistance to carbapenems, whereas the only isolate in which ISAba1 was not located near the blaOXA-23-like gene was susceptible to carbapenems. The ISAba1 sequence position of another blaOXA-23-like-positive isolate was inconclusive. The isolates exclusively carrying the blaOXA-51-like gene (n = 7) showed susceptibility to carbapenems. Conclusions: The presence of the ISAba1 sequence upstream of the blaOXA-23-like gene was strongly associated with carbapenem resistance in isolates of the A. calcoaceticus-A. baumannii complex in the hospital center studied.


INTRODUCTION
Healthcare-associated infections (HAI) are increasingly affecting public and private healthcare systems and raising not only hospital costs but also the morbidity and mortality rates of affected patients (1) (2) (3) .The bacterial genus Acinetobacter is recognized as a key HAI-causing pathogen that is associated with high mortality rates in hospital settings (2) (4) .Acinetobacter has considerable capacity to spread and develop new antibiotic resistance mechanisms (5) .Consequently, numerous HAI outbreaks caused by multidrug-resistant (MDR) representatives of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex have been reported in several countries, including Brazil (2) (5) (6) .
The diversity of the genetic elements involved in the resistance of the A. calcoaceticus-A.baumannii complex to antibiotics has spurred research on these determinants of resistance (1) (2) (4) (16) (17) (18) (19) .On the basis of the fi ndings of these studies, the present study examined the occurrence of the genetic elements bla OXA and ISAba1 in clinical isolates belonging to the A. calcoaceticus-A.baumannii complex and their association with phenotypic susceptibility to carbapenem antibiotics.

Selection of clinical isolates and ethical aspects
The study was conducted at Dona Helena Hospital, a private general hospital with 189 beds, and at the Molecular Biology Laboratory of the University of Region of Joinville [Universidade da Região de Joinville (UNIVILLE)], both located in Joinville, Santa Catarina, Brazil.Clinical isolates belonging to the A. calcoaceticus-A.baumannii complex identifi ed over 6 consecutive years, starting in March 2009, were included in the study.Isolates related to HAI (healthcareassociated infection after 48h of admission), colonization (presence of the microorganism without clinical signs of related infection), and surveillance culture upon admission (detection of colonization in patients referred from other healthcare institutions at the time of hospital admission) according to the criteria defi ned by the Hospital Infection Control Service of the hospital center studied were included in the study.Only the fi rst isolate obtained from patients with 2 or more isolates derived from the same anatomical site during the same period of hospitalization was considered for this study.All isolates were identifi ed by the Microscan Walkaway automated system (Siemens, Munich, Germany) complemented by Gram staining.
This study was evaluated and approved by the Research Ethics Committee of UNIVILLE (Opinion 788.455).

Antibiotic susceptibility analysis
The determination of the antibiotic susceptibility profi le was performed using the disc-agar diffusion method (Kirby and Bauer) (20) .The inhibition halos were interpreted according to the current recommendations of the Clinical and Laboratory Standards Institute (CLSI) (21) for each corresponding year.

Bacterial DNA preparation and evaluation
The heat shock method was used to prepare bacterial deoxyribonucleic acid (DNA) from a pure culture grown in MacConkey agar (22) .Initially, 3 colonies collected using a bacteriological loop were resuspended in 100μL of ultrapure sterile water (Milli-Q, São Paulo, Brazil).The suspension was subjected to a boiling water bath for 5 min and then cold shocked in an ice bath for 5 min.The boiling and cooling procedures were repeated, followed by centrifugation at 8,000 ×g for 10 min.The supernatant containing the bacterial DNA was evaluated through spectrophotometric readings at 260 and 280nm and then stored at -20°C.
The extracted bacterial DNA was assessed by polymerase chain reaction (PCR) to determine its viability for use in the subsequent genotypic analyses.A pair of primers specifi c to the A. calcoaceticus-A.baumannii complex [gltA-F (5′-AATTTACAGTGGCACATTAGGTCC-3′) and gltA-R (5′-GCAGAGATACCAGCAGAGATACACG-3′)] was used to amplify a single 722-bp fragment of the gltA gene encoding bacterial citrate synthase (23) .The thermocycling conditions were as follows: initial denaturation at 94°C for 3 min; 30 cycles of 94°C for 1 min, 62°C for 30 s, and 72°C for 1 min; and fi nal extension at 72°C for 10 min (23) .

Investigation of the genetic elements associated with oxacillinase production
The occurrence of the bla OXA-23-like , bla OXA-24-like , bla OXA-51-like , bla OXA-58-like , and bla OXA-143-like genes in the selected isolates was investigated using multiplex PCR with the primers outlined in Table 1 (24) (25) .The reactions were conducted in a fi nal volume of 50μL containing approximately 50-500ng of extracted DNA and a reagent mixture with 1U Platinum® Taq DNA polymerase (Invitrogen, São Paulo, Brazil), 200μM dNTPs mix (GE Healthcare, Little Chalfont, United Kingdom), 1× PCR buffer (Invitrogen), 20 pmol of each primer (DNA Express, São Paulo, Brazil), and 1.5mM of MgCl 2 (Invitrogen).The reactions were performed in an XP Cycler device (BIOER Technology, Tokyo, Japan) at the following conditions: initial denaturation at 94°C for 3 min; 30 cycles of 1 min at 94°C, 30s at 52°C, and 1 min at 72°C; and fi nal extension at 72°C for 10 min (24) .
The presence of the ISAba1 promoter sequence in the isolates and its association with the bla OXA-23-like and bla OXA-51-like genes were investigated by PCR (12) (26) (27) .The primer sequences and the sizes of the expected reaction products are outlined in Table 1.Excluding the primers (10 pmol of each primer per reaction), all other components were used in quantities identical to those described for the multiplex PCR, and the reactions were performed using the same thermocycler.The initial denaturation was performed at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 1 min, hybridization at 50°C for 1 min, and extension at 72°C for 2 min.The fi nal extension was performed at 72ºC for 5 min.The PCR products were subjected to submerged electrophoresis (TBE buffer, 10 V/cm) in a 1% agarose gel with 0.5µg/mL ethidium bromide.The expected fragments were confi rmed by exposure to ultraviolet light (300nm) in a transilluminator (MiniBis-Pro Photodocumentation System -DNR Bio-Image Systems Ltd., Jerusalem, Israel), followed by scanning.

Data analysis
A database was created using Excel 2013 (Microsoft Corp., Redmond, WA, USA) for phenotypic and genotypic data collection and analysis.Appropriate percentages were calculated, and data were analyzed descriptively.

RESULTS
Seventy-eight clinical isolates belonging to the A. calcoaceticus-A.baumannii complex derived from 60 patients (12 individuals contributed 2 or more isolates) were employed in the phenotypic and genotypic analyses established for the study.All isolates were positive for amplifi cation of the bla OXA-51-like gene fragment.These isolates were identifi ed in clinical samples from patients with HAI (66.7%), surveillance culture upon admission (15.4%), and colonization (17.9%).
The isolates were collected from bronchoalveolar lavage (38.5%), wound secretions (12.8%), rectal swabs (12.8%), blood (9%), urine (6.4%), and other types of samples (20.5%).The distribution of isolates by hospital department showed that 53.8% were derived from the intensive care unit (ICU); 29.5%, from the inpatient units; 10.3%, from the emergency room; and 6.4%, from the surgical center.The temporal evolution in relation to the sectors and types of samples corresponding to the analyzed isolates is shown in Table 2.

Genotypic evaluation
All extracted DNA samples enabled the amplifi cation of the gltA gene segment and were considered viable for subsequent genotypic analyses.Figure 2 depicts the electrophoretic pattern resulting from the PCR analysis of the main subgroups of genes encoding oxacillinases and ISAba1 in the clinical isolates of the present study.
All isolates carrying the bla OXA-23-like gene (n = 71) also had the ISAba1 promoter sequence; this sequence was located upstream from the bla OXA-23-like gene in 69 isolates.A single isolate carried ISAba1, which was not associated with the location of bla OXA-

23-like
, whereas another isolate proved inconclusive in terms of the insertion position.The ISAba1 sequence was not observed upstream of the bla OXA-51-like gene in the analyzed isolates.

Evaluation of the association between genetic markers and phenotypic resistance to carbapenems
All isolates with ISAba1 located upstream of the bla OXA-23-like gene (n = 69) showed resistance to imipenem and meropenem and were identifi ed during the 6 consecutive years of observation (93.7%, 91.7%, 100%, 91.7%, 85%, and 72.7% of the resistant isolates in each year).Conversely, the only isolate in which ISAba1 was not coupled to bla OXA-23-like was susceptible to carbapenems and was identifi ed in year 1.The isolates exclusively containing bla OXA-51-like (n = 7; 1 in year 2, 1 in year 4, 2 in year 5, and 3 in year 6) also showed full susceptibility to imipenem and meropenem.

DISCUSSION
Carbapenems are the antibiotics of choice for the treatment of infections caused by β-lactam-resistant bacteria of the A. calcoaceticus-A.baumannii complex.However, resistance to carbapenems has increased, thereby limiting the use of this class of antibiotics (15) .The present study demonstrated that the presence of the ISAba1 promoter sequence associated with the bla OXA-23-like gene in A. calcoaceticus-A.baumannii was strongly associated with resistance to carbapenem antibiotics in the hospital setting.
The species A. baumannii is the most important and representative member of the A. calcoaceticus-A baumannii complex, and it is considered the leading pathogen of the Acinetobacter genus in health institutions worldwide (10) .Small genomic differences exist among the species of this genus and have been reported by several authors; however, A. calcoaceticus, A. baumannii, A. pittii, and A. nosocomialis are closely related and are collectively termed the A. calcoaceticus-A.baumannii complex owing to the difficulty in separating them using conventional tests (16) (28) .A. baumannii species identifi cation may be confi rmed by detecting the presence of the bla OXA-51 gene because it naturally occurs in the A. baumannii chromosome (29) . This gene is intrinsic to the species and has been identifi ed in a wide collection of isolates of the A. calcoaceticus-A.baumannii complex collected from various geographic areas (13) (28) (29) (31) (32) .Thus, phenotypically identifi ed isolates that possess the bla OXA-51-like gene were considered members of the complex in the present study.Conversely, recent studies have reported the plasmid-encoded carbapenemase OXA-51 in clinical isolates of Acinetobacter nosocomialis and in other genospecies not corresponding to A. baumannii (17) (33) (34) (35) .
Acinetobacter baumannii has become particularly problematic due to its increased prevalence and resistance to carbapenem antibiotics (36) .In the present study, 88.5% (69/78) of the isolates showed resistance to carbapenems.According to the Antimicrobial Surveillance Program in Latin America, the resistance rates of Acinetobacter spp. to carbapenems increased from 6.4%, 12.6%, and 0% between 1997 and 1999 to 84.9%, 71.4%, and 50.0% between 2008 and 2010 in Argentina, Brazil, and Chile, respectively (36) (37) .A cohort study conducted in 5 ICUs of tertiary hospitals of the metropolitan area of the City of Goiânia, Goiás, Brazil, showed a high rate of A. baumannii resistance to carbapenems (73.4%), which is higher than the mean values reported by other Brazilian institutions (25-45%) (38) .
Oxacillinase production corresponding to bla OXA-23-like expression is the most common carbapenem resistance mechanism in A. calcoaceticus-A.baumannii reported in various countries, including Bulgaria, China, Iraq, Afghanistan, France, and Polynesia (39) .In the present study, 91% (71/78) of the isolates were positive for bla OXA-23-like amplifi cation.A study conducted by Carvalho et al. (17) in 10 public and private hospitals in the City of Rio de Janeiro from 2005 to 2007 showed similar results, with the detection of bla OXA-23-like in 96% of the isolates.This rate is higher than the rates observed in studies conducted in other countries, both nationwide, such as in Taiwan (21.1%), or restricted to a few local hospitals, such as in Romania (84.6%),Tunisia (82%), Greece (72.4%), and Italy (40%) (33) .
The expression of bla OXA genes might be increased by the presence of the ISAba1 sequence upstream of the enzymeencoding gene, which provides higher levels of resistance to carbapenems (14) .The presence of ISAba1 was detected in all isolates positive for bla OXA-23-like gene amplifi cation in the present study (n = 71).Furthermore, all isolates wherein ISAba1 was located upstream of the bla OXA gene (n = 69) showed resistance to carbapenems.This profi le of a strong association between ISAba1 and bla OXA-23-like was repeated during the 6 years of observation, which suggests the occurrence of cross-infection, dissemination, and long-term permanence of this resistance mechanism in the hospital center studied.The only isolate wherein ISAba1 was not located upstream of the bla OXA-23-like gene showed susceptibility to carbapenems.Thus, the presence of bla OXA-23-like without the ISAba1 promoter sequence might not be suffi cient to cause carbapenem hydrolysis, as reported by Carvalho et al. (40) OXA-51 carbapenemases encoded by intrinsic (chromosomal) genes have a weak capacity to hydrolyze carbapenems (11) (41) .The presence of ISAba1 may provide a strong promoter for the bla OXA-51-like gene, thereby increasing its expression and causing resistance (42) (43) (44) .A study conducted in the United Kingdom by Turton et al. (27) showed that carbapenem hydrolysis by isolates carrying the bla OXA-51-like gene only occurred in bacteria in which ISAba1 was upstream of the gene, suggesting that this IS acted as a promoter for the expression of this gene.Antonio et al. (26) reported that, in their study performed in hospitals of the Southeast and South regions of Brazil from 2004 to 2008, ISAba1 was not observed upstream of the bla OXA-51-like gene; furthermore, none of the isolates analyzed showed the carbapenem-resistant phenotype.The results in our study were similar because only 9% (7/78) of the isolates exclusively amplifi ed bla OXA-51-like , and none amplifi ed ISAba1 concomitantly; moreover, all these isolates were susceptible to carbapenems.This fi nding reinforces the hypothesis that the absence of ISAba1 or its presence in a region not upstream of the bla OXA-51-like gene renders a very low or nonexistent ability to hydrolyze carbapenems in A. baumannii isolates without other bla OXA genes.
The emergence of carbapenem resistance has limited the treatment of HAIs caused by A. baumannii (36) (38) .
In the present study, 41% of the isolates showed resistance to ampicillin/sulbactam, which is nearly half that of the 85.2% rate reported by Carvalho et al. (45) from 2002 to 2010 in the ICU of a public institution in the City of Teresina, Piauí, Brazil.The use of ampicillin/sulbactam has been effective for the treatment of systemic infections caused by MDR A. baumannii; however, a steady decline in susceptibility rates has been reported in recent decades (46) (47) .Furthermore, in Latin America, A. baumannii has been commonly reported as being MDR, with high rates of resistance to other antibiotics in addition to carbapenems, including ceftazidime, piperacillin/ tazobactam, and ciprofl oxacin.Thus, Latin America appears to be the highest-ranking region in the world for the prevalence of MDR A. baumannii (38) .In a study conducted using 155 A. baumannii isolates from 11 Brazilian states from 2008 to 2011, Chagas et al. (36) demonstrated that most isolates also showed resistance to more than 5 classes of antibiotics in addition to carbapenems.The highest rates were observed for ciprofl oxacin, cefepime, and piperacillin/tazobactam, with 99.4% resistant isolates, followed by ceftazidime (97.4%), sulfamethoxazole/trimethoprim (23.9%), and amikacin (11.6%); however, the isolates did display susceptibility to polymyxin B. In the present study, 88.3%, 87.2%, and 68.4% of the isolates showed resistance to ciprofl oxacin, piperacillin/tazobactam, and ceftazidime, respectively, and all analyzed isolates were susceptible to polymyxin B. However, higher rates of resistance were observed for sulfamethoxazole/trimethoprim (74.5%) and amikacin (41%).These data suggest that the use of polymyxins should be highlighted as a therapeutic option for HAIs caused by A. baumannii (48) .Polymyxin B and colistin have long been used as antibiotic agents, but they cause nephrotoxicity and neurotoxicity.Nevertheless, they are considered effective in treating infections caused by carbapenem-resistant A. baumannii (49) .Unfortunately, resistance to these antibiotics is also emerging (36) (50) .
Acinetobacter baumannii also stands out because it is an opportunistic pathogen in HAI episodes, especially in ICUs (34) .In particular, invasive mechanical ventilation -a life support method widely used in ICUs -is usually associated with nosocomial infection by A. baumannii (51) .In a study performed using 500 isolates from 30 hospitals of 10 Cuban provinces from 2010 to 2012, Quiñones et al. (37) demonstrated that the hospital unit with the highest number of cases of A. calcoaceticus-A.baumannii infection was the ICU (60.2%) and that the respiratory tract had the highest rate of positive cultures (38.6%).Godoy et al. (38) showed that the lungs were also the most common site of infection with A. baumannii among patients monitored in 5 ICUs of Goiânia (53.1% of cases).Similar results were observed in our study, in which 53.8% (42/78) of the isolates were derived from the ICU, and the majority of positive cultures were derived from bronchoalveolar lavage (66.7%; 28/42).
Acinetobacter calcoaceticus-A.baumannii colonization should be analyzed because it may precede cases of infection and contribute to the endemicity of this agent in healthcare institutions, in addition to being a key microorganism related to HAIs (52) .In the present study, 17.9% (14/78) of the isolates were related to patient colonization, a prevalence higher than the 9.4% A. baumannii colonization rate reported by Godoy et al. (38) .Surveillance cultures of patients transferred from other institutions were adopted in the hospital studied during year 3.A total of 12 cases positive for A. baumannii were confi rmed, including 7 in year 5 (6 isolates from rectal swabs and 1 from wound secretion) and 5 in the following year (4 isolates derived from rectal swabs and 1 from tissue fragments).All the cases were identifi ed within 48h of admission.
Our study had some limitations.First, the number of isolates was relatively small despite the period of 6 years covered by the analyses, and the study was performed at a single hospital center.Second, the included patients had several comorbidities, and some underwent multiple hospital admissions, which complicated the evaluation of the impact on the numbers related to colonization.Therefore, we recommend conducting similar multicenter studies involving a greater number of hospitals and evaluating the clinical and epidemiological characteristics associated with HAIs and the risk factors.Furthermore, we propose an investigation of the association between genetic profi les related to resistance and the severity and clinical outcomes of the affected patients using standardized clinical scores.Finally, we consider it pertinent to conduct the genotyping of isolates identifi ed in the hospital to examine the clonal relationship between resident strains and possible community-acquired strains, thereby highlighting possible fl aws in infection control.
In conclusion, this study provides data on the prevalence of isolates belonging to the A. calcoaceticus-A.baumannii complex carrying genetic elements associated with carbapenem resistance.The emergence and spread of variants of the bla OXA gene encoding carbapenemases and of ISs capable of increasing bla OXA expression in A. baumannii in Brazil underscore the need for the medical community and health managers to remain alert to the potential threat of outbreaks of carbapenem-resistant isolates, especially those with the ISAba1 sequence upstream of the bla OXA-23-like gene.

TABLE 1
Primers used to detect genes encoding oxacillinases and ISAba1.

TABLE 2
Distribution of clinical isolates according to sample type and hospital department over 6 years(2009-2015).