Carbapenem-Resistant Acinetobacter baumannii Recovered from Swine Manure

Abstract

Recently, concerns have been raised about the possibility of Acinetobacter baumannii transmission between animals and humans. So far, A. baumannii has been reported in animals with which people can come into contact. The presence of this pathogen in animal manure presents an equally important public health risk. In this study, we report the finding of two A. baumannii isolates in swine manure from a Croatian pig farm. Both isolates shared features with the widespread human clinical isolates: affiliation to the international clonal lineage 2 (ST-195), carbapenem, and extensive drug resistance and the plasmid-located acquired bla_OXA-23 gene. These two A. baumannii isolates survived anaerobic conditions, competition with other microorganisms, and elevated concentrations of heavy metals in the stagnant swine manure for at least 2 weeks. These findings call for bacteriological analysis and disinfection of liquid swine manure before its application as a fertilizer in traditional extensive agriculture.

Introduction

The bacterium Acinetobacter baumannii is a pathogen common in hospital-acquired and community-acquired human infections.¹ So far, we know little about the sources of A. baumannii outside of the hospital setting. Recently, however, concerns have been raised about the possibility of A. baumannii exchange between human and animals. So far, 6, 3, and 165 antibiotic-sensitive A. baumannii isolates have been confirmed in chicken, geese, and white stork nestlings, respectively.² Two hundred twenty antibiotic-sensitive isolates have been confirmed in dogs, cats, rabbits, ferrets, snakes, rats, and ducks in veterinary clinics.³

Carbapenem-resistant A. baumannii has received special attention, because carbapenems are often used as last-resort antibiotics to treat infections caused by antibiotic-resistant bacteria. In Croatian hospitals the carbapenem-resistant A. baumannii accounted for 86% of all isolates in 2016.⁴ Yet no Croatian study has investigated the presence of A. baumannii in animals to date. The rest of the world, however, reports only a few carbapenem-resistant A. baumannii isolates in animals. A single carbapenem-resistant A. baumannii was isolated from a pig with pneumonia and sepsis.⁵ Five carbapenem-resistant A. baumannii isolates were found in infected dogs and two in cats.^3,6,7

But it is not only the animals with which people come in direct contact that present a risk of infection with carbapenem-resistant A. baumannii isolates but also their feces or urine. Namely, livestock muck is often used to fertilize soil, especially in traditional extensive agriculture. The use of livestock manure as soil fertilizer for crop production is strictly prescribed by national regulations, but these do not include detection of carbapenem-resistant bacteria. Moreover, the manure is not analyzed at all in the case of private traditional crop production worldwide. If the manure contains clinically relevant A. baumannii, community-acquired¹ human infections may occur through contact with manure, fertilized soil, cultivated plants, or the food chain. Patients with community-acquired A. baumannii infections may represent the source for consequent hospital outbreaks.¹ Therefore, the presence of clinically relevant A. baumannii in livestock manure that is used to fertilize soil represents a public health concern.

The aim of this study was to look for the presence of carbapenem-resistant bacteria in manure used as fertilizer from a pig farm in Croatia. In this study, we report the first evidence of clinically relevant carbapenem-resistant A. baumannii in swine manure.

Materials and Methods

Swine manure sampling

The manure was sampled at a private pig farm in the northern continental part of Croatia in July 2017. At the farm, 730 pigs were bred for fattening using commercial food and drinking water. No signs of disease were observed during the breeding period. The manure accumulated during the swine breeding was collected in a retention basin and was used before the vegetation period as the organic fertilizer for the farmland. The sampling of manure has been done with the owner's permission 2 weeks after the swine were slaughtered. A sample of liquid swine manure from the central part of the retention basin was aseptically collected in a sterile plastic bottle and transported to the laboratory within 5 hours.

Mineralogical and chemical analyses

The manure sample was air-dried at room temperature and pulverized in agate mortar. Its mineral composition was determined with X-ray powder diffraction (XRD) using a Philips diffractometer (graphite monochromator, CuKα radiation, proportional counter). The identification of the mineral phases was based on the methods outlined by Moore and Reynolds.⁸ Selected macro- (Ca, Mg, Na, and K) and microelements (Fe, Mn, Cu, Cd, Pb, and Zn) were analyzed in triplicates with AAS (Aanalyst 700) following digestion in a mixture of nitric and perchloric acid.⁹

Bacteriological analyses

The fresh liquid manure was analyzed in technical triplicate after its suspension and dilution in sterile peptone water. Aerobically grown total heterotrophic bacteria were determined on a nutrient agar (Biolife) after incubation at 22°C for 72 h.¹⁰ The intestinal enterococci were determined as indicators of manure fecal pollution.¹¹ Membrane filters were incubated on Slanetz Bartley agar (Biolife) at 37°C for 72 h, and intestinal enterococci confirmed on bile esculin azide agar (Sigma-Aldrich) after incubation at 44°C for 4 h.¹² Carbapenem-resistant bacteria (CRB) were determined on the CHROMagar Acinetobacter medium supplemented with CR102 (CHROMagar), intended for the cultivation of clinically relevant carbapenem-resistant bacteria, after incubation at both 37°C for 72 h and 42°C for 48 h (CRB37 and CRB42, respectively). Incubation at 42°C suppresses the growth of environmental autochthonous species with intrinsic resistance to carbapenems.^13–15 Total heterotrophic bacteria, intestinal enterococci, and carbapenem-resistant bacterial counts were determined as colony forming units (CFUs), logarithmically transformed and expressed as log CFU per 1 mL of manure. The prevalence of carbapenem-resistant bacteria among total heterotrophic bacteria was calculated as (log CFU/mL _{carbapenem-resistant}/log CFU/mL _{heterotrophic}) × 100.

Identification and characterization of A. baumannii

We isolated two presumptive A. baumannii colonies from the plates on which we counted CRB42. The colonies were recultivated (42°C/24 h) on the CHROMagar Acinetobacter medium supplemented with CR102 and then on nutrient agar. A. baumannii was identified on a Vitek2 system (bioMérieux) following the routine bacteriological procedure and confirmed with matrix-assisted laser desorption ionization-time of flight mass spectrometry—MALDI-TOF MS (software version 3.0, Microflex LT; Bruker Daltonics) of the cell extracts.¹⁶

To assess the genetic relationship between the two A. baumannii isolates and the described clinical and environmental isolates, the multilocus sequence typing (MLST) of fragments of seven traditional housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) was performed according to the Oxford MLST scheme (http://pubmlst.org/abaumannii/). Fragments amplified by PCR (ProFlex™ 96-Well PCR System; Applied Biosystems) were sequenced on both strands (commercial service Macrogen Europe, The Netherlands). Raw nucleotide sequences were assembled and manually edited using the Geneious software (www.geneious.com/). The sequence type (ST) was retrieved from the A. baumannii MLST website (http://pubmlst.org/abaumannii/).

The susceptibility of the isolates to carbapenems (meropenem and imipenem), fluoroquinolones (ciprofloxacin and levofloxacin), aminoglycosides (tobramycin, gentamicin, and amikacin), penicillins/β-lactamase inhibitors (ampicillin/sulbactam), folate pathway inhibitors (trimethoprim/sulfamethoxazole), and polymyxins (colistin) was determined from the minimum inhibitory concentrations (MICs) obtained with the Vitek2 system using the AST-XN05 and AST-GN69 testing cards. Susceptibility to colistin was confirmed by broth microdilution. MICs were interpreted according to the European Committee on Antimicrobial Susceptibility Testing¹⁷ criteria for all antibiotics with defined breakpoints for Acinetobacter spp., while for ampicillin/sulbactam Clinical and Laboratory Standards Institute¹⁸ breakpoints were used. Genes of the bla_OXA lineage that encode OXA-type carbapenemases were amplified by PCR using specific primers for intrinsic bla_{OXA-51-family} and acquired bla_OXA-40-like and bla_OXA-23-like genes.^19,20 Obtained amplicons of bla_OXA genes were sequenced and edited as described for the MLST analysis. The sequences were then aligned with ClustalX 2.1.²¹ and subjected to the phylogenetic analysis using the MEGA 7 software.²² To determine if the acquired oxacillinase genes were plasmid encoded, we also isolated plasmid DNA from an overnight culture using the PureLink™ Quick Plasmid Miniprep Kit (Thermo Fisher Scientific, USA) and then amplified and analyzed the bla_OXA genes as described above. All representative sequences were deposited in the GenBank.

Results

Characterization of swine manure

The XRD showed amorphous matter as the main constituent of the swine manure with the struvite-type phosphate mineral, calcite, and quartz constituting minor mineral fractions (Fig. 1). As the manure contained a high content of potassium and sodium (Table 1) and as at least part of it was in the crystalline phase, we cannot be sure whether the sample contained pure struvite. Therefore, we defined this phase as the struvite-type phosphate mineral (d-spacings of struvite-type phosphate minerals are very similar). For example, struvite-(K) contains potassium instead of ammonium, while hazenite is the first struvite-type compound that contains two structurally distinct monovalent cations (K and Na).²³ As for the content of the selected macro- and microelements (Table 1), Cu and Zn concentrations were high; 88 and 679 mg/kg, respectively.

FIG. 1.

X-ray diffractogram of swine manure.

Table 1.

Content (in mg/kg) of Selected Macro- and Microelements in the Investigated Swine Manure

Ca	Mg	K	Na	Fe	Mn	Cu	Zn	Cd	Pb
36,541 ± 1,857	12,450 ± 490	24,490 ± 324	2,773 ± 43	1,220 ± 14	363 ± 7	88 ± 3	679 ± 6	BDL	BDL

Average values of triplicate measurements and standard deviations are given. Detection limits for Cd and Pb were 0.036 and 0.26 mg/kg, respectively.

BDL, below detection limit.

Bacteriological analyses

Being pH-neutral, the swine manure contained high counts of aerobic total heterotrophic bacteria and intestinal enterococci as indicators of fecal pollution (Table 2). Presumably environmental, intrinsically resistant CRB37 prevailed over the presumably clinically important CRB42 with acquired resistance.

Table 2.

Bacteriological Characteristics of Swine Manure

Group of bacteria	log CFU/mL (prevalence %)
Total heterotrophs	8.4 ± 0.1
Intestinal enterococci	6.6 ± 0.3
CRB37	3.1 ± 0.1 (37%)
CRB42	0.8 ± 0.3 (10%)

Average values of triplicate measurements and standard deviations are given. Prevalence of CRB among total heterotrophic bacteria was calculated as (log CFU/mL_CRB/log CFU/mL_heterotrophs) × 100. The pH value of manure was 7.51; detection limit was 1 CFU/mL.

CFU, colony forming unit; CRB, carbapenem-resistant bacteria.

The two colonies isolated from the plates intended for CRB42 counting gave the MALDI-TOF scores of 2.172 and 2.363, identifying both as A. baumannii. The MLST of the two A. baumannii isolates (named 3/1 and 3/9) showed that they belonged to the international clonal lineage 2 (IC2) within the ST-195 by the Oxford scheme (Table 3).

Table 3.

The Multilocus Sequence Typing Results, Presence of bla_OXA Lineage Genes, and Minimum Inhibitory Concentration Values of Tested Antibiotics for Two Acinetobacter Baumannii Isolates Recovered from Swine Manure

Isolate	Sequence type	Clonal lineage	Intrinsic bla_OXA	Acquired bla_OXA	MIC values of antibiotics (mg/L)
Isolate	Sequence type	Clonal lineage	Intrinsic bla_OXA	Acquired bla_OXA	MEM	IPM	CIP	LVX	TOB	GEN	AMK	SAM	SXT	CST
3/1	ST-195	IC2	OXA-66	OXA-23	≥16^R	≥16^R	≥4^R	4^R	≥16^R	≥16^R	≥256^R	16^I	≥32^R	1
3/9	ST-195	IC2	OXA-66	OXA-23	≥16^R	≥16^R	≥4^R	4^R	≥16^R	≥16^R	≥256^R	16^I	1.5	0.75

Carbapenems (MEM-meropenem, IMI-imipenem), fluoroquinolones (CIP-ciprofloxacin, LVX-levofloxacin), aminoglycosides (TOB-tobramycin, GEN-gentamicin, AMK-amikacin), penicillins/β-lactamase inhibitors (SAM-ampicillin/sulbactam), folate pathway inhibitors (SXT-trimethoprim/sulfamethoxazole), polymyxins (CST-colistin). ^R—resistant, ^I—intermediate according to European Committee on Antimicrobial Susceptibility Testing or Clinical and Laboratory Standards Institute criteria.

MIC, minimum inhibitory concentration.

Both isolates harbored the intrinsic bla_{OXA-51 family} gene (Table 3), which showed 100% identity with bla_OXA-66 sequences belonging to the OXA-51 family of oxacillinases (Fig. 2; GenBank accession number: MH010866). The isolates were carbapenem resistant, harboring the acquired bla_OXA-23 gene located on the plasmid (Table 3). The sequences of the bla_OXA-23 gene amplified from both isolates showed 100% identity with the bla_OXA-23 sequences from the clinical isolates available in GenBank (Fig. 2; GenBank accession numbers: MH010864 and MH010865). Moreover, the isolates shared the resistance to fluoroquinolones and aminoglycosides and intermediate behavior to ampicillin/sulbactam. Isolate 3/1 was also resistant to trimethoprim/sulfamethoxazole, while isolate 3/9 was sensitive. Both remained susceptible to colistin. According to the criteria for nonsusceptibility to antimicrobial categories,²⁴ both isolates could be classified as extensively drug resistant.

FIG. 2.

Unrooted phylogenetic 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-family} are marked with a black dot, while bla_{oxa-23-family} is marked with a black triangle.

Discussion

Carbapenem-resistant A. baumannii of animal origin is rarely detected using the cultivation at 37°C. In fact, Zhang et al.⁵ reported a single carbapenem-resistant A. baumannii isolate carrying the bla_NDM-1 gene among 1,293 samples obtained from pig, chicken, and duck farms. Likewise, among 323 bacterial isolates from the feces of dairy cattle only one A. baumannii intermediately resistant to ertapenem, carrying the chromosomally encoded intrinsic bla_OXA-497 inside the OXA-51-like group, was detected.²⁵ Among the 223 A. baumannii isolates from dogs and cats, only three were carbapenem resistant, carrying the bla_OXA-23 gene and belonging to IC1 and 8.³ In another study,²⁶ among the 50 rectal swabs from cows nine were found with Acinetobacter genomospecies 15TU isolates harboring the bla_OXA-23 gene. One carbapenem-resistant A. baumannii carrying the bla_OXA-23 gene localized on the chromosome and belonging to IC2 was reported from cat urinary tract infection.⁷ Low prevalence of carbapenem-resistant A. baumannii could be related to the relatively low temperature used for its isolation from animal samples. Namely, increasing the incubation temperature to 42°C seems to yield more successful isolation of A. baumannii from environmental samples.^13–15 In this study, we recovered two carbapenem-resistant A. baumannii isolates from swine manure, even though the prevalence of CRB42 was relatively low (10%). Both harbored the acquired plasmid-located bla_OXA-23 genes closely related to those found in clinical isolates. While the intrinsic chromosomally encoded carbapenem-resistance genes bla_OXA-51-like are of low epidemiological relevance, the plasmid-located bla_OXA-23 genes may be self-transferable,²⁷ which raises public health concern.

Carbapenems are not approved for the treatment of livestock anywhere in the world.²⁸ However, there is one report from China that a pig farm used different β-lactams in combination with β-lactamase inhibitors to prevent or treat infections.⁵ The farmer at the investigated Croatian pig farm denied any use of penicillin and/or β-lactamase inhibitors, which put selective pressure for OXA-23. The A. baumannii isolates from the swine manure may have received OXA-23 from the environmental progenitor of the bla_OXA-23 such as A. radioresistens.²⁹ It is also possible that carbapenem resistance in the two A. baumannii isolates developed under the influence of emerging pollutants or undefined conditions in stagnant swine manure or that carbapenem-resistant A. baumannii had been present but undetected in live pigs and excreted through feces or urine in the manure. Although we cannot pinpoint the true source of the carbapenem-resistant A. baumannii isolates, these had remained viable in the manure 2 weeks after the swine were slaughtered.

To our knowledge, no carbapenem-resistant A. baumannii isolates from swine manure have been reported so far. The two isolates recovered in this study belong to the ST-195 within IC2. A. baumannii belonging to IC2 are predominant among human clinical isolates in Europe.³⁰ In fact, they were recently found in hospitalized patients, untreated hospital wastewater, urban sewage, and river water in Zagreb,³¹ as well as in the soil at an illegal dump site near Rijeka in Croatia.³² This stresses the clinical importance of A. baumannii recovered from the swine manure.

Of note the recovered A. baumannii isolates in our study tolerated the elevated concentrations of Cu and Zn in the stagnant anaerobic swine manure. However, these concentrations (88 mg Cu/kg and 679 mg Zn/kg) were below the legal limits for organic manure in Croatia. It seems that high Cu and Zn concentrations are common in raw swine manure. A Chinese study³³ reported 151.11 and 538.29 mg/kg, respectively, whereas an Austrian study³⁴ reported 282 and 1,156 mg/kg, respectively. Clearly, Cu and Zn may accumulate in swine manure if there is a source such as animal feed, in which both are widely used to stimulate growth.³⁵ Heavy metals possess potential antibacterial activity, which depend on the binding of metal ions to the manure particles and its consequent leaching. Obviously, detected concentrations of Cu and Zn did not express antibacterial activity against the A. baumannii recovered from the manure. The observation is consistent with the report where A. baumannii was recovered from soil containing even 2,719 mg Zn/kg.³²

Moreover, the two A. baumannii isolates survived competition with other bacteria present in the manure, such as intestinal enterococci, whose counts were comparable to those reported in fresh manure in France.¹¹ This confirms that intestinal enterococci are reliable indicators of pig fecal pollution that could persist in the manure for at least 2 months.¹¹

Our findings provide an insight into what kind of environmental factors are tolerated by A. baumannii, which has not received much attention so far. Not only can A. baumannii survive anaerobic conditions and a competitive environment but also it tolerates high concentrations of heavy metals. The finding calls for bacteriological analysis and disinfection (alkaline, thermal, biological, chemical, or physical)³⁶ of liquid swine manure before it is applied as fertilizer in traditional extensive agriculture.

Footnotes

Acknowledgments

This work has been supported by the Croatian Science Foundation (project no. IP-2014-09-5656). The authors thank S. Kazazic, Rudjer Boskovic Institute for MALDI-TOF MS identification.

Compliance with Ethical Standards

This research fully complies with Ethical Standards applicable for this Journal and the relevant national and international ethics related rules and professional codes of conduct.

Disclosure Statement

No competing financial interests exist.

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