Occurrence of Carbapenemase-Producing Klebsiella pneumoniae in Bat Guano

Abstract

The aim of this study was to screen for the presence of carbapenemase-producing Enterobacteriaceae (CPE) isolates from bat guano in Bejaia, Algeria. Guano samples (n = 110) were collected in Aokas's cave, Bejaia, Algeria, between March and May 2016. Samples were plated on MacConkey agar supplemented with ertapenem (0.5 mg/L) and vancomycin (32 mg/L). The isolates were identified and antimicrobial susceptibility was determined using disk diffusion method. Carbapenemase, extended spectrum β-lactamases, plasmid-mediated AmpC, and plasmid-mediated quinolone resistance genes were studied using PCR and sequencing. Clonal relatedness was studied using multilocus sequence typing (MLST). Two CPE isolates were identified as Klebsiella pneumoniae. PCR and sequencing identified the blaOXA-48 in one K. pneumoniae strain (CS34) and blaKPC-3 in the other strain (CS63). K. pneumoniae CS63 was found to carry bla_TEM-1 and aac(6′)-Ib genes. The MLST showed that K. pneumoniae CS63 was assigned to ST512, whereas K. pneumoniae CS34 belonged to ST1878. This is the first description of CPE from bats' guano.

Introduction

The most important mechanism of carbapenem resistance in Enterobacteriaceae is the production of carbapenemases, mainly KPC, VIM, NDM, and OXA-48.¹ Of these, KPC-type carbapenemase-producing Klebsiella pneumoniae (KPC-KP) is a great concern.² KPC-KP emerged in 1996 in the United States³ and since then has disseminated globally, becoming endemic in some countries.⁴ The rapid and efficient dissemination of KPC-KP has mostly been caused by the clonal expansion of clonal complex CC258.⁵

OXA-48 was first identified in K. pneumoniae, in 2001, in Turkey. Since then OXA-48-producing Enterobacteriaceae increased dramatically, especially in Mediterranean countries.⁶

Antimicrobial resistance is an urgent global health challenge in human and veterinary medicine. Wild animals are not directly exposed to clinically relevant antibiotics; however, antibacterial resistance in wild animals has been increasingly reported.⁷

Bats are unique among mammals in their ability to fly and cover long distances during seasonal migrations.⁸ They contribute to the regulation of insect populations in their habitats, pollination of flowers and dispersal of seeds.⁹ However, they receive increasing attention in infectious disease studies, because of their association with emerging infections as vectors of zoonotic pathogens.¹⁰

With 25 identified species, bats represent the second largest group of mammals in Algeria after rodents.¹¹

In 1988, only one report describing the antibiotic resistance profile of Enterobacteriaceae strains isolated from wild mammals (including bats) in Indonesia was published¹² and since then very few studies were reported. So far, no studies have described β-lactam resistance in Enterobacteriaceae isolated from bats. To our knowledge, no report described the production of carbapenemases in bat guano. The aim of this study was to screen for the presence of carbapenemase-producing Enterobacteriaceae (CPE) in bat guano at Aokas's cave of Bejaia, Algeria.

Materials and Methods

Microbiological analysis

Between March and May 2016, bats' guano pellets were collected using sterile swabs at the Aokas's cave, located 27 km east of Bejaia city, Algeria. Samples were then transported to the laboratory.

Samples were incubated for 18 hr at 37°C in nutrient broth supplemented with 0.5 mg/L of ertapenem (ETP) and 32 mg/L of vancomycin. Cultures were then inoculated by streaking 100 μL of the suspensions onto MacConkey agar plates containing ETP (0.5 mg/L) and incubated at 37°C for 24 hr.¹³

Suggestive colonies of Enterobacteriaceae were further identified by classical biochemical tests and mass spectrometry MALDI-TOF system (IVD MALDI Biotyper, BrukerBiospin SAS, Wissembourg, France).¹⁴

Antimicrobial susceptibility testing

Antibiotic susceptibility was determined on Mueller–Hinton agar using the standard disk diffusion procedure as described by European Committee on Antimicrobial Susceptibility Testing (EUCAST).¹⁵ Seventeen beta-lactam antibiotics were tested, including amoxicillin-clavulanic acid (AMC) (20/10 μg), ticarcillin (TIC) (75 μg), ticarcillin-clavulanic acid (TCC) (75/10 μg), piperacillin (PIP) (30 μg), piperacillin-tazobactam (TZP) (30/6 μg), cefotaxime (CTX) (30 μg), cefotaxime-clavulanic acid (CCTX) (30/10 μg), ceftazidime (CAZ) (30 μg), ceftazidime-clavulanic acid (CCAZ) (30/10 μg), cefoxitin (FOX) (30 μg), aztreonam (ATM) (30 μg), cefepime (FEP) (30 μg), cefepime-clavulanic acid (CFEP) (30/10 μg), cefpirome (CPO) (30 μg), imipenem (IMP) (10 μg), ETP (10 μg), temocillin (TEM) (30 μg), and 12 non-beta-lactam antibiotics, including gentamicin (GM) (10 μg), tobramycin (TM) (10 μg), amikacin (AN) (30 μg), nalidixic acid (NA) (30 μg), norfloxacin (NOR) (10 μg), ciprofloxacin (CIP) (5 μg), ofloxacin (OFX) (5 μg), tetracycline (TET) (30 μg), trimethoprim (TMP) (5 μg), sulfonamides (SSS) (200 μg), trimethoprim/sulfamethoxazole (SXT) (1,25/23,75 μg), fosfomycin (FOS) (200 μg), and chloramphenicol (C) (30 μg) (BioRad, Marnes La Coquette, France).

Isolates classified as resistant or intermediate to ETP by disk diffusion were further analyzed by determining the minimum inhibitory concentrations (MICs) of IMP, ETP, meropenem, and TEM by E test (AB bioMérieux, Marcy l′Etoile, France). MICs were interpreted according to EUCAST 2018 breakpoints.

Phenotypic detection of carbapenemases

The carbapenem inactivation method (CIM) test was performed as described by Van der Zwaluw et al.,¹⁶ using Escherichia coli ATCC 25922 as a negative control and K. pneumoniae U2A1977 (NDM-1) as a positive control.

Detection of antibiotic resistance genes

Total DNA was extracted using the automate Nuclisens Easymag R. V2.0 method (bioMérieux, Marcy l′Etoile, France).

Multiplex PCRs were performed to screen for the presence of the carbapenemase genes (bla_KPC, bla_VIM, bla_IMP, bla_NDM, bla_OXA-23-like, bla_OXA-24-like, bla_OXA-58-like, and bla_OXA-48-like) and plasmid-mediated AmpC (pAmpC) genes (CMY-2/BIL/LAT, CMY-1/MOX, DHA, FOX, ACC, and ACT/MIR) as previously described.^17,18

Standard PCRs were performed for detection of extended spectrum β-lactamase (ESBL)-encoding genes (bla_TEM, bla_CTX-M, bla_SHV, and bla_GES) as described previously.^18,19

The qnr, qepA, and oqxAB genes were screened by real-time PCR, as previously described.^20,21 Pyrosequencing method was used for the detection of aac(6′)-Ib-cr and aac(6′)-Ib genes.²²

Standard PCR targeting mcr-1 was performed as described by Liu et al.²³

Negative and positive controls were used in all PCRs.

All PCR products were sequenced and the sequencing results were compared with reported sequences available in GenBank.

Molecular typing

Multilocus sequence typing (MLST) was performed targeting seven conserved K. pneumoniae housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB), according to Pasteur schemes available on the Institute Pasteur MLST website.²⁴

Conjugation and transformation experiments

Conjugation experiments were performed using E. coli J53 (sodium azide resistant) as the recipient.²⁵ Transconjugants were selected using brain heart infusion (BHI) agar plate supplemented with ETP (1 μg/mL) and sodium azide (200 μg/mL). When no transconjugants were obtained, transformation assays were performed as described By Gharout-Sait et al.¹⁷

Furthermore, MICs and PCR amplifications targeting carbapenemases genes were also performed on the transconjugant.

PCR-based replicon typing

Plasmid incompatibility groups were determined using PCR-based replicon typing.²⁶

Results

Of the 110 bat guano samples tested, 2 carbapenem K. pneumoniae resistant isolates were obtained. The CIM test was positive for the two strains.

The disk diffusion method showed that the two isolates displayed various antimicrobial susceptibility patterns (Table 1). The MICs evaluated for the CS63 isolate confirmed the resistance to carbapenems; however, the MICs for CS34 isolate confirmed the low level of resistance to ETP only and TEM (Table 1).

Table 1.

Characteristics of Carbapenemase-Producing Klebsiella pneumoniae Isolates and Their Transconjugants Form Bat Guano in Bejaia, Algeria

Code (carbapenemase)	Resistance pattern ^a	MICs (mg/L)				Others genes	MLST	Replicon typing
Code (carbapenemase)	Resistance pattern ^a	IMP	Meropenem	ETP	TEM	Others genes	MLST	Replicon typing
CS34 (OXA-48)	AMC, TIC, TTC, TZP, FOX, TEM, ETP, NAL, TMP, SSS, C	0.5	0.5	1	256	oqxAB	ST1878	IncL/M
TrCS34^b	AMC, TIC, ETP, TTC	0.5	0.25	0.5	256			IncL/M
CS63 (KPC-3)	AMC, TIC, TTC, TZP, FOX, CTX, CCTX, CAZ, CCAZ, FEP, CFEP, CPO, ATM, TEM, ETP, IMP, TM, AN, NAL, NOR, OFX, CIP, TET, TMP, SXT, SSS, C	8	32	32	32	bla_TEM, aac(6′)Ib, oqxAB	ST512	IncFIIAsIncFII1KIncFII2K

Intermediate or resistant to against tested antibiotics.

Transconjugant.

AMC, amoxicillin-clavulanic acid; TIC, ticarcillin; TCC, ticarcillin-clavulanic acid; PIP, piperacillin; TZP, piperacillin-tazobactam; CTX, cefotaxime; CCTX, cefotaxime-clavulanic acid; CAZ, ceftazidime; CCAZ, ceftazidime-clavulanic acid; FOX, cefoxitin; ATM, aztreonam; FEP, cefepime; CFEP, cefepime-clavulanic acid; CPO, cefpirome; IMP, imipenem; ETP, ertapenem; TEM, temocillin; TM, tobramycin; AN, amikacin; NA, nalidixic acid; NOR, norfloxacin; CIP, ciprofloxacin; OFX, ofloxacin; TET, tetracycline; TMP, trimethoprim; SSS, sulfonamides; SXT, trimethoprim/sulfamethoxazole; C, chloramphenicol; MLST, multilocus sequence typing.

Molecular characterization by PCR and DNA sequencing showed that the carbapenemase-producing isolates harbored the bla_OXA-48 gene (CS34) and bla_KPC-3 gene (CS63). In addition, K. pneumoniae CS63 was found to carry bla_TEM-1 and aac(6′)-Ib genes. None of the isolates was found to carry ESBL, pAmpC, mcr-1 and plasmid-mediated quinolone resistance genes.

The clonal relationship of isolates analyzed by MLST showed that the OXA-48-producing K. pneumoniae isolate was assigned to sequence type ST1878 and KPC-3-producing K. pneumoniae isolate belonged to ST512 (Table 1).

Transfer of bla_OXA-48 was successfully obtained. In addition, the transconjugant (TSCS34) showed the same MICs as the donor. An IncL/M-type plasmid was found in this transconjugant.

Nonetheless, neither transconjugants nor transformants were obtained for CS63 isolate.

Discussion

The emergence and global dissemination of CPE constitute a major public health concern.²⁷

In addition to the NDM-1-producing Salmonella reported in black kites (Milvus migrans) in Germany,²⁸ OXA-48-producing Enterobacteriaceae from wild boars²⁹ and white storks were reported in Algeria.³⁰ Other CPE were also reported from wild animals, Bhardwaj et al. reported the isolation of NDM-5-producing E. coli strains from a tiger in India,³¹ IMP-producing Enterobacteriaceae were isolated in silver gulls in Australia.³² Vittecoq et al. reported the isolation of VIM-1-producing E. coli in gulls from southern France.³³

Thus, we report in this study the first isolation of CPE from bat guano.

bla_OXA-48-like genes have been most often found in K. pneumoniae.³⁴ Since the first description of the OXA-48 carbapenemase in Turkey, the enzyme has been extensively reported in many parts of the world, particularly in the Mediterranean area. In Algeria, it has been detected in human infections, in the hospital environment, in river water, in companion animals, in fresh vegetables, and wildlife.³⁴

In our study, OXA-48-producing K. pneumoniae strain was assigned to ST1878. This ST has been described only in the Klebsiella Pasteur MLST database. The strain was isolated from premature baby (rectal swab) in Neonatal Intensive Care Unit at University Medical Center Groningen, Netherlands.²⁴

The bla_OXA-48 gene carriage by a frequently reported IncL/M-type plasmid³⁵ demonstrated the cross contaminations between wild animals and environment. The IncL/M plasmids are commonly identified among environmental and clinical isolates.³⁶ The high transfer efficiency of the epidemic IncL/M plasmid to any enterobacterial species is the reason for the successful spread of bla_OXA-48.³⁴

KPC-KP has spread rapidly and extensively in some countries. KPC-2 and -3 are the most prevalent variants, whereas K. pneumoniae is their predominant host species.³⁷ In Algeria, one study described a KPC-3-KP ST512 isolated from cerebrospinal fluid of child.³⁸ In this study, KPC-3-producing K. pneumoniae belonged to ST512. This ST was observed in human infections. The KPC-3-producing K. pneumoniae ST512 clone has emerged as a successful new lineage, mainly responsible for the dissemination of KPC-type carbapenemases on the global scale.³⁹

To our knowledge, this is the first description of CPE isolated from bat guano and the first description of KPC-3-producing Enterobacteriaceae isolated from wild animals.

It has been reported that the diet to be a primary factor to define the gut microbiome of bats.⁴⁰ Noticeably, Algerian species are insectivorous.¹¹ Of note, insectivorous bats act as natural pest control agents in a single night and save millions of dollars in agricultural pesticides.⁴⁰ The air above bodies of water is usually rich in insects and, therefore, constitutes an important feeding habitat for many species of bats.⁴¹ Thus, the ingestion of flies by insectivores could be a route of transmission of antibiotic-resistant bacteria. Furthermore, insects can act as potential vectors for the spread of resistant bacteria to different environments.⁴² Consequently, they play a role in the spread of antibiotic-resistant bacteria between humans and animals. Recent studies have shown that the fly gut provides an appropriate environment for carriage of antimicrobial resistant bacteria and horizontal transfer of plasmids carrying antibiotic resistance genes.⁴³ Resistant bacteria in flies often share the same genotypes with bacteria from humans and animals when their habitats overlap. In addition, degrading insects can contribute to environment contamination with antimicrobial-resistant bacteria.⁴² Environmental compartments can also serve as reservoirs for β-lactam resistance genes, and a variety of bla genes have been identified in bacteria derived from fecal sludge and lagoon water from dairy farms, water, or sediment from aquaculture areas, wastewater treatment plants and surface waters.⁴⁴ Antimicrobial-resistant bacteria were found in different type of surface waters.⁴⁵ In fact, rivers and sea are exposed to discharges from various sources, receiving microbial contaminants from industrial, agricultural, and domestic sources.^44,46

Few studies have reported carbapenemase producers among Enterobacteriaceae isolated in water environments around the world. Bejaia is a coastal city known for its dense hydrographic network. Enterobacteriaceae producers of bla_OXA-48 were isolated in water samples from the Soummam River, which is contaminated with urban, industrial, and agricultural discharges.⁴⁷

The cave's environment is humid and can be contaminated by bacteria of human and animal origins. Another risk factor for the spread of carbapenemase-producing bacteria throughout the wild environment can be the wild birds; they can easily transmit these strains to water course and other environment sources.²⁹ Wild birds are considered to be a reservoir of carbapenemase-encoding genes.³⁰ Thus, transmission of carbapenem-resistant bacteria may be associated with several environmental factors as birds feeding habits, the habitat they visit to feed, and their habitat. Wild animals might play a vital role in the worldwide spread of clinically relevant pathogens or resistance genes.⁷

Limitations of our study are limited size of the samples and the method of sampling. It is interesting to perform cloacal samples.

In conclusion, the present results, and those reported in similar works, show that wild animals could play a significant role in the dynamic diffusion of antimicrobial resistance genes. Thus, continued surveillance of multiresistant bacteria in wild animals is warranted.

Footnotes

Acknowledgment

The authors thank Janick Madoux from CHU Reims for her technical assistance.

Disclosure Statement

No competing financial interests exist.

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