Pan Drug-Resistant Environmental Isolate of Acinetobacter baumannii from Croatia

Introduction

Multidrug-resistant (MDR) Acinetobacter baumannii has emerged as one of the most common nosocomial pathogens worldwide, with the ability to survive under a wide range of environmental conditions, and to persist for extended periods of time on biotic and abiotic surfaces.^1,2 A. baumannii is considered an equivalent to methicillin-resistant Staphylococcus aureus and, therefore, called “Gram-negative MRSA.” ³ Increasing antimicrobial resistance leaves very few therapeutic options, especially in carbapenem-resistant Acinetobacter isolates that are reported worldwide. These isolates are susceptible only to polymyxins—peptide antibiotics that are not routinely used because of earlier reports about toxicities. Polymixin-resistant A. baumannii occurred almost exclusively in patients who had received colistin for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infections. ⁴ Pan drug-resistant clinical isolates that demonstrate resistance to all antimicrobial agents, including polymyxins, have also been reported in the literature, making treatment of these infections extremely difficult and in some cases impossible with fatal outcome.^4,5

In Croatia, the first clinical carbapenem-resistant isolate of A. baumannii was recovered in 2002 in southern part of Croatia. ⁶ Over the period of 7 years, carbapenem resistance in clinical isolates of A. baumannii did not exceed 30%, but since 2009, incidence of carbapenem resistance drastically increased, especially in two biggest clinical centers in northern and southern parts of Croatia. ⁷ Carbapenem resistance in A. baumannii has rapidly spread throughout Croatia, and in 2014 nonsusceptibility to carbapenems reached 82%. ⁸ This resulted in a change of treatment options for infections caused by MDR A. baumannii, foregrounding ampicillin/sulbactam and colistin as the only therapeutic choice. Recently published data confirmed the occurrence of environmental carbapenem-resistant isolates of A. baumannii related to clinical isolates in untreated and treated wastewater in Croatia. ⁹ However, to date there is no report on the occurrence of either clinical or environmental pan drug-resistant isolates of A. baumannii in Croatia. Here we report the first evidence of pan drug-resistant A. baumannii in treated wastewater in Zagreb, Croatia.

Materials and Methods

The composite 24 hr sample of effluent wastewater was collected on September 9, 2015 at the secondary type municipal wastewater treatment plant of the City of Zagreb, Croatia. ¹⁰ The isolation of A. baumannii was performed at 42°C/48 hr on CHROMagar Acinetobacter (CHROMagar) supplemented with CR102 and 15 mg/L cefsulodin sodium salt hydrate (Sigma-Aldrich). Identification of isolate was performed by routine bacteriological techniques, Vitek 2 system (BioMerieux), and MALDI-TOF MS (software version 3.0, Microflex LT; Bruker Daltonics) on cell extracts. ¹¹ Molecular identification was performed by amplification of a fragment of rpoB gene encoding RNA polymerase β-subunit by using rpoB+1627/rpoB-2231 primer pair. ¹² Amplified fragments were sequenced, edited, and analyzed as described previously. ⁹

Susceptibility testing was done by using Vitek 2 system and E-test, and minimum inhibitory concentrations (MICs) were interpreted according to EUCAST and CLSI criteria^13,14 for all antibiotics with defined breakpoints for Acinetobacter spp., except tigecycline where breakpoints for Enterobacteriaceae were used. The presence of genes of bla_OXA lineage that encode OXA-type carbapenemases was searched by multiplex polymerase chain reaction (PCR) with specific primers for bla_OXA-51-like, bla_OXA-40-like, bla_OXA-23-like, and bla_OXA-58-like genes. ¹⁵

Results and Discussion

The isolate of A. baumannii named EF7 was recovered from 0.5 ml of the secondary treated municipal wastewater of the City of Zagreb. By phenotypical analyses and Vitek 2 system, the isolate was determined as A. calcoaceticus–A. baumannii complex. MALDI-TOF MS analysis gave the score value of 2.150, which indicates the secure genus and probable species identification. Phylogenetic analysis of the ∼600 bp fragment of rpoB gene confirmed the identification of isolate as A. baumannii with 100% sequence identity (KX468061) to the reference sequence from GenBank (CP007535).

The isolate was resistant to all tested antibiotics (penicillins/β-lactamase inhibitors, carbapenems, fluoroquinolones, aminoglycosides, folate pathway inhibitors, and polymyxins) except intermediately susceptible to tetracyclines (Table 1), thus categorized as pan drug resistant. ¹⁶ The isolate remained intermediately susceptible to minocycline (MIC: 8 mg/L), which is not at market in Croatia, and tigecycline (MIC: 2 mg/L), which is recently present at the market in Croatia and is accepted as part of the combined treatment of MDR Acinetobacter infections. Environmental isolate EF7 showed very similar antibiotic resistance profile to pan drug-resistant clinical isolate of A. baumannii recovered in 2013 in Germany ⁵ with the exception of tigecycline resistance in the German isolate. The levels of antibiotic resistance of isolate EF7 were comparable with those of the clinical isolates of A. baumannii in Croatia,^7,17 with exception that no clinical isolates resistant both to ampicillin/sulbactam and colistin were registered. Ampicillin/sulbactam and colistin were used in recommended therapy in Zagreb's hospitals in recent years, including the period of wastewater sampling. It is possible that the pan drug resistance was developed in hospital and isolate EF7 was discharged into sewage system.

Multiplex PCR and sequencing confirmed the presence of both intrinsic chromosomally located bla_OXA-51-like gene and acquired plasmid-located bla_OXA-23-like gene. Sequences of bla_OXA-51-like (KX468062) and bla_OXA-23-like (KX468063) genes showed 100% identity with sequences from clinical isolates available in GenBank (Fig. 1), which suggest the potential origin of isolate EF7 in hospitalized patients or hospital wastewaters. ¹⁸ Untreated hospital wastewaters in Zagreb are directly discharged into sewage system, which is collected and treated at investigated central wastewater treatment plant. Removal of carbapenem-resistant bacteria and A. baumannii in the secondary wastewater treatment plant is moderate. ¹⁰ Thus, certain number of A. baumannii is released through the effluent wastewater into the natural recipient Sava River. In aquatic environmental conditions MDR A. baumannii has the potential to multiply and survive up to 50 days. ¹⁰ Therefore, the viable pan drug-resistant A. baumannii recovered from effluent wastewater could be easily spread through the natural water body, which could represent an environmental reservoir of this emerging pathogen. Moreover, since the plasmid-located bla_OXA-23-like gene in isolate EF7 may be self-transferable, ¹⁹ the effluent wastewater represents a potential epidemiological reservoir of the carbapenem resistance gene. The impact of secondary treated municipal wastewaters on the occurrence of community-acquired A. baumannii infections outside the hospital environment should be further investigated.

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FIG. 1.

Unrooted phylogentic tree (NJ method, number of differences) constructed on the basis of bla_OXA genes encoding OXA-type carbapenemases. GenBank accession numbers are given next to the name of each strain. Gene sequences of bla_OXA-51 type are marked with a black dot and of bla_OXA-23 type with a black triangle.