Current Microbiology (2022) 79:385

https://doi.org/10.1007/s00284-022-03092-0

Genomic Characterization of Twelve Lytic Bacteriophages Infecting

Midgut Bacteria of Aedes aegypti
Osvaldo López‑Cuevas1 · Jean P. González‑Gómez1 · José R. Aguirre‑Sánchez1 · Bruno Gomez‑Gil2 ·
Edith H. Torres‑Montoya3 · José A. Medrano‑Félix4 · Célida I. Martínez‑Rodríguez1 ·
Nohelia Castro‑del Campo1 · Cristóbal Chaidez1

Received: 24 February 2022 / Accepted: 15 October 2022 / Published online: 3 November 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Mosquito-borne diseases such as malaria and dengue are global severe public health threats. Due to the lack of efficient
control methods, alternative approaches to decreasing arboviral transmitted diseases are prioritized to reduce morbidity and
mortality in every endemic region. Mosquito midgut bacteria play an essential role in physiological development, fitness, and
the arthropods´ vectorial capacity. Bacteriophages are viruses that infect bacteria and are considered a promising biocontrol
method by eliminating midgut microbiota that plays an essential role in mosquitoes´ health. Here, we isolate and identify 22
bacteria from mosquito´s midgut belonging to the genera Mesobacillus, Enterobacter, Klebsiella, Microbacterium, Micrococ-
cus, Pantoea, Serratia, and Staphylococcus, mainly. Twelve phages with lytic activity against Enterobacter, Klebsiella, and
Pantoea were also isolated. All 12 phages showed a double-stranded DNA genome, ranging from 36,790 to 149,913 bp, and
were taxonomically classified as members of the Drexlerviridae family, Molineuxvirinae, Studiervirinae, and Vequintaviri-
nae subfamilies. Open reading frames associated with phage structure, packing, host lysis, DNA metabolism, and additional
functions were predicted in all 12 phage genomes, while tRNAs were predicted in five phage genomes. In addition, the life
cycle was predicted as virulent for the 12 phages, and no antibiotic resistance, virulence, allergenic, or lysogenic genes were
found in either genome. These findings suggest that the 12 phages have biocontrol potentials; however, it is necessary to
elucidate specific bacterial host’s roles and then the phages' ability to serve as effective vector control.

Introduction

The pace at which pathogens transmitted by arthropod-vec-

* Cristóbal Chaidez
chaqui@ciad.mx
1
Laboratorio Nacional Para La Investigación en Inocuidad
Alimentaria (LANIIA), Centro de Investigación en
Alimentación Y Desarrollo, A.C. (CIAD), Carretera a
Eldorado Km 5.5, Campo El Diez, 80110 Culiacán, Sinaloa,
México
2
Centro de Investigación en Alimentación Y Desarrollo,
A.C. (CIAD), Unidad Mazatlán en Acuicultura Y Manejo
Ambiental, 711, Mazatlán, Sinaloa, México
3
Laboratorio de Conservación de La Fauna Silvestre, Facultad
de Biología, Universidad Autónoma de Sinaloa. Avenida
de Las Américas Y Boulevard Universitarios S/N,
80010 Culiacán, Sinaloa, México
4
Investigadoras e Investigadores Por México-Centro
de Investigación en Alimentación Y Desarrollo A.C,
Coordinación Regional Culiacán. Laboratorio Nacional Para
La Investigación en Inocuidad Alimentaria, Sinaloa, México
tors advance is significant. Half of the world’s population
lives at risk of arthropod-vector diseases [1]. Tropical and
subtropical areas, known to be endemic for mosquito-borne
diseases, are affected the most due to a global warming cli-
mate and increasing travel, migration, and international trade
[2]. Vector-borne diseases cause more than 17% of all infec-
tious diseases, with a death number of over 700,000 per year.
Even though malaria and other mosquitoes-borne parasites
lead the morbidity and mortality data, Dengue fever is the
most widely distributed viral disease transmitted by Aedes
mosquitoes around the globe, with an incidence of more than
3.9 billion cases and 40,000 deaths in more than 129 coun-
tries [1]. Recently, the WHO has declared an urgent public
health priority to improve better vector control to stop the
spread of vector-borne diseases [3]. Undoubtedly, early use
of insecticide has resulted in the best intervention method
to mitigate the advance of the mosquitoes and the pathogens

13
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385 Page 2 of 14
they carry; however, the emergence of insecticide-resistance
mosquitoes´ strains has resulted from the exacerbated use of
temephos, malathion, novaluron, and other chemical insec-
ticides [4]. Also, exposure to insecticides has consequences
on human health and beneficial species such as bees; thus,
effectively developing novel strategies to reduce transmis-
sion of pathogens by insect vectors is a priority [5, 6].
Substantial efforts are underway to exploit genetic infor-
mation provided by the genome-sequencing project and
design practical tools to develop new vector control meth-
ods. For example, induced reproduction of sterile mosquito
populations for their release into the environment and para-
transgenesis; both are in experimental stages to date [7].
In parallel, studies on the basic insect midgut microbiome
are helping unravel processes contributing to its diversity,
regulation, and the insect´s ability to transmit deadly human
pathogens [8, 9]. Mosquitoes’ midgut microbiota plays an
essential role in their health. They participate in vital physi-
ological functions such as metabolism, reproduction, and
immunity. They also play crucial roles in the nutrient provi-
sion that is limited or not provided by the diet [10]. Ramirez
et al. [11] suggested that antimicrobial peptides produced
by intentionally inoculated bacteria (Pantoea sp., Proteus
sp., and Paenibacillus sp.) on the midgut of Aedes aegypti
prevented the increasing Dengue virus load on mosquitoes
by eliciting innate mosquito immunity. Contrarily, Serra-
tia odorifera could increase Dengue virus infections in A.
aegypti by producing a polypeptide blocking the prohibitin;
this last is a molecule present on the midgut of female A.
aegypti participating in immune response mechanisms [12].
They suggested that the elimination of Serratia could repre-
sent a significant advance for the reduction of vector-borne
diseases.
Because some bacteria participate positively in physi-
ological processes such as the reproduction and develop-
ment of the mosquito, these bacteria may be considered
“targets” for vector control purposes. On the other hand,
other bacteria species can interrupt the vectorial capacity
of some mosquito species, and they could be considered as
“non-target” for vector control purposes. Thus, it is crucial
to identify specific bacterial species' roles and selectively
eliminate those bacteria participating in mosquito´s fitness.
In most experimental investigations, antibiotics were used
to eliminate mosquito´s midgut bacterial species [11, 12].
However, antibiotics eliminate various bacterial species, so
it is challenging to identify the roles of specific bacteria spe-
cies in mosquito life.
Bacteriophages are viruses infecting specifically bacteria
and Archaea. These viruses are highly selective for genera
or species and replicate by two alternative cycles: lytic and
lysogenic life cycles. In the lytic cycle, phages infect bac-
teria, leading to new phage particles production and bac-
teria degradation (lysis). During the lysogenic cycle, the
13
O. López‑Cuevas et al.
genetic material of the phage may integrate into the bacterial
genome, a process that does not lead to bacterial lysis [13].
To our best knowledge, the most successful approach for
selective elimination of bacterial species from any microbial
consortium is by lytic phage administrations. Phage therapy
in humans is the best example in this context and has been
used for a long time [14]; however, phage administration for
selective biocontrol against bacterial species in arthropod
midgut has been poorly studied. Zhang et al. [15] demon-
strated that bacteriophage administration on housefly larvae
gut successfully reduced up to 90% of Pseudomonas aerugi-
nosa populations. They also demonstrated that phage admin-
istration every 24 h produced changes in the gut bacterial
composition and affected the larvae health. They concluded
that deficiency in one symbiotic gut bacteria by selective
elimination with phages leads to changes in the intestinal
microbial composition in housefly larvae, and it also pro-
duces adverse effects on the development of the insect.
Experimental bacterial elimination by phage administra-
tions could unveil its participation in biological mosquitos’
functions. Additionally, phage could be used as a biological
control method by reducing/eliminating specific bacterial
species playing a particular role in mosquito’s bodily func-
tions. Therefore, the objective of the present study was to
isolate and characterize bacteriophages with lytic activity
against culturable bacteria from the midgut of female Aedes
aegypti mosquitoes captured in the capital city of Sinaloa in
northwestern Mexico.
Material and Methods
A suction device was built to capture adult stage mosquitoes.
Wild mosquitoes were collected in selected neighborhoods
from Culiacan, Sinaloa, Mexico. The selection criteria for
the sampling points were taken to favor the chances of find-
ing adult mosquitoes (as cool, damp, and dark places). Mos-
quitoes were captured by suction while at rest or in flight
and transferred to the laboratory in refrigeration 8–10 °C.
Five samples were taken in different city sectors, and every
sample consisted of at least ten adult mosquitoes.
Mosquitoes Identification and Selection
Female Aedes aegypti specimens were identified using a
stereomicroscope and based on the taxonomic keys pro-
posed by Rueda [16]. Briefly, adults of Aedes aegypti spec-
imens were selected based on their dark coloration, acute
abdomen, white rings at the base of the tarsal, tibial, and
femur segments of the legs, and a pattern of white bands
on the mesothorax. Male specimens were distinguished
from females by their characteristic feathery antennae
Genomic Characterization of Twelve Lytic Bacteriophages Infecting Midgut Bacteria of Aedes…
and longer palps. Those specimens that did not meet the
taxonomic characteristics of female Aedes aegypti were
discarded.
Middle Bowel Dissection
Autoclaved sterile materials were used, and utensils and
surfaces were ethanol sanitized in all the experiments. First,
the mosquitoes were anesthetized by cold (4 °C for 5 min).
Next, they were washed with 70% ethanol for 5 min and then
rinsed three times in phosphate-buffered solution (PBS, pH
7.4), preventing external contamination. From the last rinse,
plantings were carried out in tryptic soy agar (TSA) medium
to corroborate exterior sanitation of the mosquitoes. Next,
the specimens were transferred to a slide-mounted under
the stereoscope by adding a drop of PBS (by stabbing the
mosquito’s chest with a dissecting pin). Then, the mosqui-
to’s abdomen was gently removed, and the middle intestine
recovered [17]. Finally, ten intestines were transferred to
a 1.5-mL microtube containing 250 µL of sterile PBS and
macerated for 30 s with a sterile pestle until homogenized
[12].
Isolation of Culturable Bacteria
From the homogenate previously obtained, serial dilutions
­(10–1–10–5) were made, and aliquots of 100 μL were plated
in duplicate on tryptic soy agar medium (TSA). The inocu-
lated samples were incubated at 37 °C for 48 h.
Differential colonial characteristics (color, size, shape,
opacity, margin, elevation, and viscosity) were consid-
ered for their selection and purification. Once the bacteria
were purified, Gram staining was performed to observe the
arrangement and morphology of the isolates. Finally, the iso-
lated bacteria were preserved in Glycerol (25%) and stored
at − 20 °C for further analysis.
Bacterial DNA Extraction
Preserved bacteria were reactivated in tryptic soy broth
(TSB) and incubated at 37 °C for 24 to 48 h. First, the bac-
terial culture was centrifuged at 14,000 × g for 5 min, dis-
carding the supernatant and recovering the bacterial pellet.
Next, the bacterial pellet was resuspended in 600 µL of lysis
buffer of the Wizard® Genomic DNA Purification kit (Pro-
mega®, USA) and processed for DNA extraction, according
to the manufacturer’s instructions. Finally, the DNA was
rehydrated in 100 µL of DNA rehydration solution incubat-
ing at 65 °C for one hour and then stored at − 20 °C for
further analysis.
Page 3 of 14 385
PCR Amplification of 16S rRNA and Bacterial
Identification
The extracted genetic material was analyzed in a NanoDrop
2000c instrument (Thermo Fisher Scientific, Delaware,
USA) to verify DNA concentration and purity; subsequently,
the integrity of the DNA was checked by 1% agarose gel
electrophoresis. Next, PCR amplification of the 16S rRNA
was performed using universal primers, forward 27F (5′-
AGA GTT TGA TCM TGG CTC AG -3′), and reverse
1492R (5′- TAC GGY TAC CTT GTT ACG ACT T -3′).
PCR reactions consisted of 1X buffer GoTaq® Green Mas-
ter Mix (Promega®, USA), 3 mM M ­ gCl2, 400 μM each
dNTP’s, 1 µM each primer, 1U Taq DNA Pol, template DNA
(1 μL/100 ng), and sterile nanopure water to a final volume
of 25 μL. The PCR conditions were as follows: an initial
denaturation step at 95 °C for 5 min; followed by 35 cycles
of a denaturation step at 95 °C for 35 s, primers alignment
at 56 °C for 35 s and an extension at 72 °C for 90 s; a final
step of 10 min at 72 °C was included, and the reaction was
stabilized at 4 °C. The amplified PCR product was approxi-
mately 1500 bp.
The quality and concentration of the amplicons were
verified by 1% agarose gel electrophoresis. The amplified
samples were sequenced at the Macrogen company in South
Korea (https://d​ na.m
​ acrog​ en.c​ om/). The sequences obtained
were aligned in the EZBioCloud database (https://​www.​
ezbio​cloud.​net/​ident​ify), and bacterial identification was
performed based on a more than 90% similarity percentage.
Isolation of Lytic Bacteriophages Infecting Aedes
aegypti Midgut Bacteria
Sampling
From several neighborhoods from Culiacan, Sinaloa, Mex-
ico, 14 samples (12 from stagnant or drain water, one from
bovine feces, and one from midgut Aedes aegypti) were
taken at places nearby the origin of the previously captured
mosquitoes. Samples were transferred under refrigeration
to the Lab for processing within six hours after sampling.
Enrichment
Preserved bacteria obtained at the previous stage were
individually grown in TSB for 24 h at 37 °C. Next, bac-
terial pools were made and independently inoculated with
the samples (in a proportion of 5/1) and incubated under
aerobic conditions at 37 °C for 18–24 h. After incubation,
samples were centrifuged at 13,800 × g for 10 min at 4 °C;
the supernatant was recovered and filtered through 0.22 µm
sterile nitrocellulose membranes, obtaining the lysates for
further analysis [18].
13
385 Page 4 of 14
Bacteriophage Detection, Isolation, Purification,
and Propagation
Every lysate previously obtained was individually chal-
lenged against isolated bacteria by the spot double over-
lay technique. Lysates showing lytic activity were selected
for phage isolation and purification. First, one sensitive
bacterial strain to lysates was selected and grown in TSB
for 24 h at 37 °C. Next, the double overlay technique
was performed, and after 24 h incubation, well-isolated
plaques were selected based on their size and clarity and
then transferred to 1.5-mL microtubes containing 1 mL
of sterile phosphate-buffered solution. The double over-
lay technique and recovery of individual plaques were
repeated four consecutive times to obtain unique and puri-
fied phages.
Bacteriophage propagation was done by the double over-
lay technique, as Carey-Smith et al. [19] proposed. Next,
6 mL of sterile buffered phosphate solution was added to
each Petri dish and allowed to rest for two hours with fre-
quent manual shaking. Afterward, the soft layer was recov-
ered using a sterile cell scraper. The final eluate was centri-
fuged at 8,500 × g for 10 min at 4 °C, and the supernatant
was then filtered through 0.45-µm sterile nitrocellulose
membranes and verified for phage presence. Finally, phage
lysates were stored at 4 °C, protected from light.
Bacteriophage Concentration and Titration
40 mL of the bacteriophage stock was taken and centrifuged
at 40,000 × g for two hours. The supernatant was carefully
decanted, and the pellet was resuspended in 10 mL of sterile
buffered phosphate solution and filtered through a 0.22-µm
sterile nitrocellulose membrane. The bacteriophage titer was
determined by preparation of decimal dilutions ­(101–1010),
and the double agar overlayer technique was performed as
described by Ackermann [13]. Dilutions at which 30–300
plaques could be counted were used to determine the phage
concentration using the following formula: PFU/mL = Num-
ber of plaques × 10 × Reciprocal of the counted dilution.
Molecular Characterization of Bacteriophages
Viral DNA Extraction
Five milliliters of the concentrated phage suspension (c.a.
1 × ­1010 PFU/mL) was added with 2 U of each DNase I/
RNase A (Sigma-Aldrich, USA), and then incubated at 37 °C
for 30 min. Then, the phage DNA extraction was done using
the SDS-proteinase K protocol proposed by Sambrook and
Russell [20]. DNA concentration and purity were verified
13
O. López‑Cuevas et al.
on a NanoDrop 2000c (Thermo Scientific, USA), and its
integrity was electrophoresed as described previously.
Genome Sequencing
The DNA libraries were prepared using the Nextera XT
Library Preparation Kit (Illumina, San Diego, CA, USA) and
quantified using a Qubit 2.0 fluorometer (Thermo Fisher Sci-
entific, Waltham, MA, USA). The genome sequencing was
performed with the Illumina MiniSeq platform (2 × 150 bp
paired-end protocol, 300 cycles). Raw reads were trimmed
by fastp v0.19.5 [21] and de novo assembled using SPAdes
v3.15.3 [22], both with default settings, resulting in a single
contig per phage genome.
Bioinformatic Analysis
The open reading frames (ORFs) were identified by phano-
tate v1.5.0 [23] and functionally annotated by performing
a kmer-based search against the CoreSEED and FigFams
using the RASTtk pipeline according to Brettin et al. [24].
The ORFs were manually curated using Geneious v9.1.8 to
resolve overlapping calls and the start/stop codons valida-
tion. The tRNA presence was identified through tRNAscan-
SE [25] and ARAGORN [26]. The virulence signatures were
screened using Victors [27] and VFDB [28], and signatures
of antibiotic resistance were screened using CARD [29] and
NDARO (http://​www.​ncbi.​nlm.​nih.​gov/​patho​gens/​antim​
icrob​ial-​resis​tance/). The phage lifestyles were predicted
using the AI-driven software platform PhageAI v0.10.0
[30], and HostPhinder v1.1 (https://​cge.​cbs.​dtu.​dk/​servi​ces/​
HostP​hinder/) was used to predict possible hosts for each
phage. Additionally, allergen proteins were explored using
the Allergen Sequence Search on Allergen Online website
(http://​www.​aller​genon​line.​com/​datab​asefa​sta.​shtml). The
comparison between phages clustered in the same taxonomic
rank was performed using EasyFig v2.2.2 [31]. Finally, the
intergenomic similarities among complete viral genomes
were calculated through the VIRIDIC web tool [32] with
blastn. All bioinformatics tools were used with default set-
tings unless otherwise specified.
Results
Isolation and Identification of Culturable Bacteria
From Midgut Aedes aegypti
Twenty-two bacterial isolates were obtained. The most prev-
alent group was Gram-positive cocci (10 isolates), followed
by Gram-negative bacilli (8 isolates), and finally, Gram-
positive bacilli (4 isolates) (Table 1).
Genomic Characterization of Twelve Lytic Bacteriophages Infecting Midgut Bacteria of Aedes… Page 5 of 14 385

Table 1  Aedes aegypti midgut bacterial isolates identified by 16S rRNA sequences
Sample name GenBank acc. No Seq. length (bp) Morphology Next Related Type Strain Type Strain Acc. No Similarity to
and Gram Type Strain (%)
staining

M1A OP492053 980 Coccus G + Staphylococcus haemolyticus NR_113345.1 97.95

M1B OP492054 1002 Coccus G + Micrococcus yunnanensis NR_116578.1 99.90
M1C OP492055 965 Bacilli G− Pantoea deleyi NR_116114.1 98.81
M1D OP492056 482 Bacilli G− Klebsiella pneumoniae NR_037084.1 94.00
M1E OP492057 1002 Bacilli G− Enterobacter hormaechei subsp. NR_126208.1 99.30
xiangfangensis
M1F OP492058 960 Bacilli G− Enterobacter hormaechei subsp. NR_126208.1 98.23
xiangfangensis
M2A OP492059 485 Bacilli G− Pantoea stewartii subsp. indolo- NR_104928.1 99.09
genes
M2B OP492060 1018 Coccus G + Micrococcus aloeverae NR_134088.1 99.71
M2C OP492061 892 Bacilli G + Microbacterium hibisci NR_158048.1 98.33
M2D OP492062 992 Bacilli G− Pantoea dispersa NR_116797.1 98.89
M2E OP492063 934 Bacilli G− Serratia rubidaea NR_024644.1 99.68
M2F OP492064 412 Coccus G + Staphylococcus warneri NR_025922.1 92.94
M2G OP492065 670 Bacilli G + Bacillus cereus NR_115526.1 98.96
M2H OP492066 1013 Bacilli G + Mesobacillus foraminis NR_042274.1 98.92
M2I OP492067 1016 Coccus G + Staphylococcus haemolyticus NR_113345.1 98.92
M2J OP492068 968 Bacilli G− Mesobacillus subterraneus NR_104749.1 99.69
M3A OP492069 962 Coccus G + Staphylococcus petrasii NR_118450.1 98.96
M3B OP492070 989 Coccus G + Staphylococcus warneri NR_025922.1 100.00
M3C OP492071 998 Coccus G + Staphylococcus warneri NR_025922.1 98.68
M3D OP492072 951 Coccus G + Staphylococcus warneri NR_025922.1 98.84
M3E OP492073 945 Coccus G + Staphylococcus hominis NR_036956.1 99.26
M3F OP492074 674 Bacilli G + Priestia aryabhattai NR_164882.1 98.38

BLASTN analysis against rRNA_typestrains/16S_riboso-
mal_RNA database of the isolates subjected to 16S rRNA
sequencing identified the following bacterial genera: Meso-
bacillus, Enterobacter, Klebsiella, Microbacterium, Mic-
rococcus, Pantoea, Serratia, and Staphylococcus, mainly
(Table 1). Staphylococcus genus was predominant in the
isolates and was present in all samples, followed by Meso-
bacillus, Enterobacter, and Pantoea genera. A diversity of
17 bacterial species belonging to 8 genera was obtained.
Isolation of Lytic Bacteriophages Infecting Bacteria
From the Midgut of Aedes aegypti
Twelve bacteriophages were isolated from drain water and
stormwater streams (Table 2), which showed lytic activ-
ity against six of the 22 bacterial isolates in the previous
stage. Sensitive bacteria to isolated bacteriophages were
Enterobacter xiangfangensis, Klebsiella pneumoniae, Pan-
toea deleyi, Pantoea dispersa, and Pantoea stewartii subsp.
indologenes; all Gram-negative bacilli, belonging to the
Enterobacteriaceae family (Table 2). The lysate belonging
to the mosquito sample showed no lytic activity against any
bacterial isolates, and of the isolated bacteriophages, none
could lyse the different Staphylococcus species or any other
Gram-positive bacteria isolated in this investigation.
Genome Analysis of Bacteriophages
The genetic material of all bacteriophages analyzed con-
sists of double-stranded DNA with a genome size ranging
between 36,790 and 149,913 bp, with a Guanine-Cytosine
(GC) content ranging 50.2–51.7%, as shown in Table 3.
The Open Reading Frames (ORFs) ranged from 55 to 322,
depending on the genome size of phages. Table 3 sum-
marizes the number of predicted ORFs for isolated bacte-
riophages. The phages were named based on their genomic
characteristics according to the Bacterial and Archaeal
Viruses Subcommittee (BAVS) of the International Commit-
tee on Taxonomy of Virus (ICTV) recommendations [33].
Bacteriophage lifestyle prediction by the PhageAI pro-
gram suggests that the twelve bacteriophages follow a lytic
cycle as a replication strategy. Additionally, the HostPhin-
der database predicted the host for each of the phages.
We found 5 (vB_PdeP_F1M1C, vB_PdeP_F2M1C,

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385 Page 6 of 14 O. López‑Cuevas et al.

Table 2  Isolation and lytic Bacteriophage Bacterial strains

effect of bacteriophage against
isolated bacteria from midgut Pantoea Klebsiella Enterobacter Enterobacter Pantoea Pantoea
Aedes aegypti deleyi pneumoniae xiangfangensis xiangfangensis stewartii dispersa

vB_PdeP_F1M1C + − − − − +
vB_KvaS_F1M1D − + − − − −
vB_ExiM_F1M1E − − + + − −
vB_PdeP_F2M1C + − − − − −
vB_KvaS_F2M1D − + − − − −
vB_ExiM_F2M1E − − + + − −
vB_KvaP_F4M1D − + − − − −
vB_ExiM_F4M1E − − + + − −
vB_PdeP_F5M1C + − − − − +
vB_KvaP_F5M1D − + − − − −
vB_ExiM_F5M1E − − + + − −
vB_PdiM_F5M2A − − − − − +

+ : lysis zone production

−: no lysis zone production

Table 3  Phages genomes characteristics

Phage Genome size (bp) ORFs tRNA %GC Life cycle Assigned Family (Fam- Host prediction
ily; Subfamily)

1 Pantoea phage vB_ 38,645 55 0 50.7 Virulent (97.48%) Autographiviridae; Salmonella enterica
PdeP_F1M1C Studiervirinae
2 Klebsiella phage vB_ 50,039 86 0 50.2 Virulent (64.50%) Drexlerviridae Klebsiella pneumoniae
KvaS_F1M1D
3 Enterobacter phage 149,828 321 17 50.6 Virulent (95.96%) Myoviridae; Vequinta- Cronobacter sakazakii
vB_ExiM_F1M1E virinae
4 Pantoea phage vB_ 39,424 57 0 50.7 Virulent (97.54%) Autographiviridae; Salmonella enterica
PdeP_F2M1C Studiervirinae
5 Klebsiella phage vB_ 49,144 82 0 50.5 Virulent (71.40%) Drexlerviridae Klebsiella pneumoniae
KvaS_F2M1D
6 Enterobacter phage 149,913 322 17 50.6 Virulent (95.62%) Myoviridae; Vequinta- Cronobacter sakazakii
vB_ExiM_F2M1E virinae
7 Klebsiella phage vB_ 45,969 61 0 51.7 Virulent (99.02%) Autographiviridae; Salmonella enterica
KvaP_F4M1D Molineuxvirinae
8 Enterobacter phage 149,912 321 17 50.6 Virulent (95.62%) Myoviridae; Vequinta- Cronobacter sakazakii
vB_ExiM_F4M1E virinae
9 Pantoea phage vB_ 36,790 56 0 50.2 Virulent (95.98%) Autographiviridae; Salmonella enterica
PdeP_F5M1C Studiervirinae
10 Klebsiella phage vB_ 45,703 60 0 51.7 Virulent (98.94%) Autographiviridae; Salmonella enterica
KvaP_F5M1D Molineuxvirinae
11 Enterobacter phage 149,913 322 17 50.6 Virulent (95.53%) Myoviridae; Vequinta- Cronobacter sakazakii
vB_ExiM_F5M1E virinae
12 Pantoea phage vB_ 149,913 321 17 50.6 Virulent (96.02%) Myoviridae; Vequinta- Cronobacter sakazakii
PdiM_F5M2A virinae

vB_KvaP_F4M1D, vB_PdeP_F5M1C, and vB_KvaP_
F5M1D), 5 (vB_ExiM_F1M1E, vB_ExiM_F2M1E,
vB_ExiM_F4M1E, vB_ExiM_F5M1E, and vB_PdiM_
F5M2A), and 2 (vB_KvaS_F1M1D, and vB_KvaS_
F2M1D) phages associated with Salmonella enterica,
Klebsiella pneumoniae, and Cronobacter sakazakii,
correspondingly. All results exhibited reliable coverage
(≥ 0.24) except for vB_KvaP_F4M1D, and vB_KvaP_
F5M1D, which had poorly reliable coverage (≤ 0.1). It
was also determined that the 12 phage genomes analysis
revealed no genes codifying for virulence or antibiotic

13
Genomic Characterization of Twelve Lytic Bacteriophages Infecting Midgut Bacteria of Aedes…
resistance in any bacteriophages. Moreover, no putative
allergen proteins were found for any of the genomes.
According to genomic analysis, the twelve bacterio-
phages were grouped into four taxonomic ranks: fam-
ily Drexlerviridae (vB_KvaS_F1M1D and vB_KvaS_
F2M1D), subfamily Molineuxvirinae (vB_KvaP_F4M1D,
and vB_KvaP_F5M1D), subfamily Studiervirinae (vB_
PdeP_F1M1C, vB_PdeP_F2M1C, and vB_PdeP_F5M1C),
and subfamily Vequintavirinae (vB_ExiM_F1M1E, vB_
ExiM_F2M1E, vB_ExiM_F4M1E, vB_ExiM_F5M1E, and
vB_PdiM_F5M2A).
Characteristics of Phages Belonging
to the Drexlerviridae Family
Klebsiella phage vB_KvaS_F1M1D and Klebsiella phage
vB_KvaS_F2M1D were grouped in the family Drexlerviri-
dae within the genus Webervirus according to the criteria
Fig. 1  Genomic analysis of phages belonging to the Drexlerviridae
family. A Comparison of Drexlerviridae phage genomes. Color scales
represent the percentage nucleotide identity between regions obtained
Page 7 of 14 385
established by the ICTV for genera demarcation [34], show-
ing > 70% nucleotide identity of the complete genome length
with previously reported phages belonging to this genus
(Fig. 1B). The predicted ORFs encode proteins associated
with DNA metabolism, structure, lysis, packaging, and addi-
tional modules (Fig. 1A). Both phages do not possess a DNA
polymerase, indicating that their replication depends on the
host´s enzymes.
Characteristics of Phages Belonging
to the Molineuxvirinae Subfamily
Klebsiella phage vB_KvaP_F4M1D and Klebsiella phage
vB_KvaP_F5M1D were grouped in the Autographiviridae
family, creating a new genus together with the Proteus
phage PmP19 and Klebsiella phage vB_KpPFBKp16
(> 79% intergenomic distance) within the Molineuxviri-
nae subfamily (Fig. 2B). The predicted ORFs encode
through blastn B Heatmap of intergenomic similarity of Drexlerviri-
dae phages calculated through VIRIDIC
13
385 Page 8 of 14
Fig. 2  Genomic analysis of phages belonging to the Molineuxvirinae
subfamily. A Comparison of Molineuxvirinae phage genomes. Color
scales represent the percentage nucleotide identity between regions
proteins associated with DNA metabolism, structure,
lysis, packaging, and additional modules (Fig. 2A).
Characteristics of Phages Belonging
to the Studiervirinae Subfamily
Pantoea phage vB_PdeP_F1M1C, Pantoea phage vB_
PdeP_F2M1C, and Pantoea phage vB_PdeP_F5M1C
were also grouped in the Autographiviridae family, within
the Teetrevirus genus, in the Studiervirinae subfamily,
according to the comparative genomic analysis (Fig. 3B).
In addition, the ORFs were predicted to encode proteins
associated with DNA metabolism, structure, lysis, pack-
aging, and additional modules (Fig. 3A).
13
O. López‑Cuevas et al.
obtained through blastn B Heatmap of intergenomic similarity of
Molineuxvirinae phages calculated through VIRIDIC
Characteristics of Phages Belonging
to the Vequintavirinae Subfamily
Enterobacter phage vB_ExiM_F1M1E, Enterobacter
phage vB_ExiM_F2M1E, Enterobacter phage vB_ExiM_
F4M1E, Enterobacter phage vB_ExiM_F5M1E, and
Pantoea phage vB_PdiM_F5M2A were grouped in the
Myoviridae family, within the Certrevirus genus, Vequin-
tavirinae subfamily, according to the comparative genomic
analysis (Fig. 4B). The predicted ORFs encode proteins
associated with DNA metabolism, structure, lysis, packag-
ing, and additional modules (Fig. 4A).
Genomic Characterization of Twelve Lytic Bacteriophages Infecting Midgut Bacteria of Aedes…
Fig. 3  Genomic analysis of phages belonging to the Studiervirinae
subfamily. A Comparison of Studiervirinae phage genomes. Color
scales represent the percentage nucleotide identity between regions
Discussion
Based on the culturable bacteria isolated from the midgut
of female Aedes aegypti in our investigation, it is well
documented that all these isolated bacterial genera are fre-
quently distributed in diverse environments (plants, water,
soil, or as normal biota of human skin). Recent research
proposes that mosquitoes acquire their intestinal bacterial
biota inherited from their mothers (vertical transfer) or by
horizontal transfer when acquired directly from nature,
mainly from the places they feed [35].
Bacillus, Enterobacter, Klebsiella, Pantoea, and Ser-
ratia genus have been frequently isolated from Aedes
aegypti mosquitoes and other mosquito species. Bacteria
can maintain a stable association with their host insect,
and they possibly play essential roles in the mosquito´s
lifestyle [11, 36].
Page 9 of 14 385
obtained through blastn B Heatmap of intergenomic similarity of
Studiervirinae phages calculated through VIRIDIC
In the present study, we found Serratia rubidaea, a
member of the Enterobacteriaceae family. Some species
of the Serratia genus, as Serratia marcescens and Serratia
odorifera, were reported as ordinary residents of mosqui-
toes’ intestinal biota, where they can participate in the
lysis of red blood cells and acidify sugars, both essential
functions for mosquitoes metabolism as they can obtain
the necessary nutrients for oviposition and fitness [12]. On
the other hand, Gaio et al. [37] determined Serratia spp.
and Enterobacter spp. were predominating in the intestine
of Aedes aegypti mosquitoes captured in Brazil and dem-
onstrated that digestion and egg production were nega-
tively affected by eliminating them through antibiotics.
Also, it has been discovered that Serratia odorifera makes
Aedes aegypti mosquitoes more susceptible to becoming
infected by the Dengue virus in Pune, India [12].
13
385 Page 10 of 14
Fig. 4  Genomic analysis of phages belonging to the Vequintavirinae
subfamily. A Comparison of Vequintavirinae phage genomes. Color
scales represent the percentage nucleotide identity between regions
Interestingly, bacteria of the Staphylococcus genus were
the most dominating in our investigation. This bacterial
genus was previously reported in Aedes albopictus mosqui-
toes captured in India, predominating in more than 60% of
the isolates [36].
Although Staphylococcus species live as commensals in
different environments, such as animal or human skin and
oral cavity, they also have been frequently isolated from
water, soil, or food, demonstrating its ability for adaptation.
Furthermore, an essential characteristic of Staphylococcus
is that they are hemolysin producers; therefore, it could be
inferred that, like the Serratia species, Staphylococcus could
participate in blood digestion and, thus, the production of
eggs and fitness in Aedes aegypti [37].
Pantoea species can also be environmentally or vertically
acquired since they colonize the mosquitoes’ sexual organs
13
O. López‑Cuevas et al.
obtained through blastn B Heatmap of intergenomic similarity of
Vequintavirinae phages calculated through VIRIDIC
[38]; however, contrary to Serratia spp. or Staphylococcus,
it was discovered that Pantoea produces bacteriocins affect-
ing pathogens, acting as part of the immune system of Aedes
aegypti mosquitoes captured in Panama [11].
The 22 isolated bacteria were used as hosts for bacterio-
phages isolation from various sources. The twelve isolated
phages showing lytic activity on some of the isolated bacte-
ria (as described in Table 2) were subject to genomic charac-
terization because the absence of virulence, antibiotic resist-
ance, and allergenicity genes is crucial to use bacteriophages
as a biological control method. Therefore, because all twelve
phages had the lytic replication genetic machinery, and no
undesirable genes were present, these findings are suitable
for considering these phages for controlling essential target
bacteria from mosquitoes midgut. In this sense, the Codex
Alimentarius Commission developed guidelines for the
Genomic Characterization of Twelve Lytic Bacteriophages Infecting Midgut Bacteria of Aedes…
evaluation of the possible allergenicity of new proteins in
food or additives; where they recommend a bioinformatics
search using nucleotide or amino acid alignments and sug-
gest that to indicate the possibility of cross-reactivity, the
coincidences in the percentage of identity are at least 35% in
segments of at least 80 amino acids. Therefore, because no
allergen coincidences were found in the analysis that meets
the Codex Alimentarius criteria, it is concluded that they
cannot generate cross-reactivity so that isolated bacterio-
phages can be considered as allergen-free, a desirable char-
acteristic for use. It is of interest considering that, although
these phages could be used on mosquitoes midgut bacterial
control, these phages could be scattered to the environment,
and eventual contact with humans could be highly possible.
With relation to the taxonomic classification of phages
infecting klebsiella species (Klebsiella phage vB_KvaS_
F1M1D and Klebsiella phage vB_KvaS_F2M1D), these
were grouped in the Drexlerviridae family consisting of
T1-like phages that predominantly infect the genus Escher-
ichia; however, phages infect the genera Cronobacter,
Enterobacter, Klebsiella, Pantoea, and Shigella have also
been isolated [39]. T1-like phages show a Siphovirus mor-
phology with an icosahedral head of about 60 nm and a
non-contractile tail of about 150 nm [40]. The comparative
genomic analysis of these phages suggests that in Weber-
virus phages, DNA packaging takes place through headful
packaging using the pac-site [41].
Both phages do not possess a DNA polymerase, indicat-
ing that their replication depends on the host’s enzymes.
The lysis module of these phages consists of three
enzymes: holin, endolysin, and spanin, which are directly
related to the lysis cassette of the T1-like-phages. Holins
and endolysins are essential for host lysis; holins control
the length of the infective cycle for lytic phages, while
endolysins are muralytic enzymes that degrade the cell wall
[42]. Spanins are bacteriophage lysis proteins responsible
for disrupting the outer membrane, the final step of Gram-
negative host lysis [43]. Both phages showed high homology
with the Klebsiella TSK1 phage, which was able to reduce
the growth of K. pneumoniae in addition to reducing their
biofilms (85–100% biomass) [41], suggesting the antibiofilm
activity in the closely related phages isolated in this study.
Klebsiella phage vB_KvaP_F4M1D and Klebsiella phage
vB_KvaP_F5M1D were grouped in the Molineuxvirinae
subfamily of the Autographiviridae family. The Autographi-
viradae family consists of podovirus with a small icosahe-
dral head attached to a short tail, and all encode a large sin-
gle subunit RNA polymerase responsible for the mid and late
transcription [44]. Bacteriophages belonging to Molineux-
virinae are T7-like viruses with six genera reported at the
date, according to the previously reported Molineuxvirinae
phages, consisting of a linear genome with short-direct ter-
minal repeats and redundant ends [45].
Page 11 of 14 385
In addition, these viruses have an ORF encoding the Ocr
protein, which mimics the DNA, blocking the DNA-binding
groove of Type I DNA restriction/modification enzymes as
experimentally demonstrated by Roberts et al. [46]. Both
phages, vB_KvaP_F4M1D and vB_KvaP_F5M1D, encode
two proteins that form part of the ejectosome complex that
degrade the bacterial cell wall forming a pore in the inner
membrane allowing the DNA translocation from the viral
capsid into the cytoplasm of the host [47]. Furthermore, both
phages showed high homology with the Klebsiella phage
vB_KpPFBKp16, which showed a latency period of 10 min
and a burst size of 113 PFU/cell, besides reducing the optic
density of K. pneumoniae culture in killing-challenge assays
[45].
Pantoea phages vB_PdeP_F1M1C, vB_PdeP_F2M1C,
and vB_PdeP_F5M1C were also grouped in the Autographi-
viridae family, within the Teetrevirus genus, in the Studi-
ervirinae subfamily. Bacteriophages belonging to the Tee-
trevirus genus are T3-like viruses showing a podovirus
morphology and around 50.8% GC.
The lytic module consists of holin, endolysin, and spa-
nin enzymes; these qualities are similar to phages of the
Drexlerviridae family. Furthermore, Studiervirinae phages
encode proteins of the ejectosome complex, similar to those
present in Molineuxvirinae phages. The additional module
of these phages contains some proteins increasing the proba-
bility of successful bacteriophage infection. Firstly, the three
phages encode an inhibitor of dGTP triphosphate hydrolase,
allowing T3-like phages to promote productive infection
since dGTPase plays an essential role in cell survival and
DNA replication [48]. Secondly, all three phages encode
a silencing suppressor. Gene silencing is often referred to
as an antiviral defense mechanism producing siRNAs tar-
geting viral genomes for degradation. Many viruses encode
silencing suppressor proteins which function to block the
production of siRNAs or the ability of siRNAs to reach their
targets [49]. Finally, these phages also encode a RecBCD
inhibitor. In bacteria, enzymes RecBCD are helicase–nucle-
ase complexes responsible for initiating homologous recom-
bination from double-stranded DNA breaks (DSBs). This
activity underpins many key DNA transactions, including
DSB repair, phage restriction, and conjugal or transductional
recombination. Thus, the RecBCD complex inhibitors help
protect the phage DNA from degradation [50]. The previ-
ously reported Klebsiella phage KPP-5 showed high homol-
ogy with the phages isolated in this study. KPP-5 infects 19
multidrug-resistant K. pneumoniae strains and has a short
latent period of 25 min and a burst size of 236 PFU/cell [51],
suggesting the bacteriolytic potential of phages belonging to
the Teetrevirus genus.
Finally, Enterobacter phages vB_ExiM_F1M1E, vB_
ExiM_F2M1E, vB_ExiM_F4M1E, vB_ExiM_F5M1E,
and Pantoea phage vB_PdiM_F5M2A were grouped in the
13
385 Page 12 of 14
Myoviridae family, within the Certrevirus genus, Vequin-
tavirinae subfamily. Phages belonging to the Vequintaviri-
nae subfamily are characterized by an icosahedral head, a
contractile tail, and a bundle of thin and flexible lateral tail
fibers that bind host surface glycans and glucose of the outer
LPS, which triggers tail contraction and DNA injection [52].
The lytic module of these phages consists of two hydro-
lases targeting cell wall and peptidoglycan, participating at
the initial stage of phage infection to penetrate the host cell
wall during injection of their genetic content [53]. Interest-
ingly, these phages possess many tRNA sequences, although
their production has not been confirmed. Lee et al. [54] sug-
gest an extra supply of tRNAs can confer certain advantages
to phage replication, as the acceleration of phage proteins
translation during the lytic cycle. Moreover, tRNAs found in
Cronobacter phage CR3 showed different codon preferences
concerning their host, suggesting that phage tRNAs could
play a role in translating phage mRNA but not host mRNA
[55]. Besides, the five phages encoded a tRNA nucleotidyl-
transferase. tRNA-nucleotidyltransferases are fascinating
and unusual RNA polymerases responsible for synthesizing
the nucleotide triplet CCA at the 3′-terminus of tRNAs. As
the CCA end represents an essential functional element for
aminoacylation and translation, these polymerases (CCA-
adding enzymes) are vital in all superior organisms but
unusual in viral genomes [56]. The previously reported
Cronobacter phage PBES 02 showed > 75% intergenomic
similarity through the VIRIDIC algorithm with the Vequin-
tavirinae phages isolated in this study, as shown in Fig. 4.
PBES 02 removed contaminating Cronobacter sakazakii
from broth infant formula and showed a latent period of
30 min and a burst size of 250 PFU/mL [54], suggesting the
potential suitability of the Certrevirus phages as biocontrol
agents.
The authors highlight insights that could help to eluci-
date two paradigms. First, isolating and characterizing lytic
bacteriophages for specific bacteria from mosquitoes midgut
could help identify which bacteria play essential roles in
the mosquito´s development and fitness. Second, once the
essential bacteria in the mosquito’s life have been identified,
bacteriophage cocktails could be prepared and applied in the
mosquito’s living environments for an indirect biocontrol of
Aedes aegypti and other mosquito´s species.
Conclusion
16S rRNA homology analysis showed 17 out of 22 isolated
bacteria belonging to the genera Mesobacillus, Enterobac-
ter, Klebsiella, Microbacterium, Micrococcus, Pantoea,
Serratia, Bacillus, Priestia, and Staphylococcus. The most
dominant intestinal isolates of female Aedes aegypti mos-
quitoes were Staphylococcus. Our results also accomplished
13
O. López‑Cuevas et al.
the genomic characterization of 12 bacteriophages, showing
lytic activity against Enterobacter, Klebsiella, and Pantoea
genera. In addition, these phages do not carry genes cod-
ing virulence, allergenicity, or antibiotic resistance factors;
therefore, these phages are suitable as biocontrol agents.
First, however, it is necessary to characterize the effect that
reducing these bacterial genera produces on mosquito fit-
ness and then formulate a phage cocktail for halting their
vectorial capacity.
Acknowledgements The authors thank MSc. Eduardo Heriberto López
Guerrero for his technical assistance.
Author Contributions OL-C: Conceptualization, Methodology, Writ-
ing—Original draft, Formal analysis. JPG-G: Conceptualization, For-
mal analysis, Methodology, Visualization. JRA-S: Formal analysis,
Methodology, Data Curation. BG-G: Methodology, Validation, Writ-
ing—review & editing. EHT-M: Methodology, Writing—review &
editing. JAM-F: Methodology, Formal analysis, Writing—review &
editing. CIM-R: Project administration, Writing—review & editing.
NC-dC: Resources, Methodology, Investigation, Writing—review &
editing. CC: Conceptualization, Funding acquisition, Validation, Writ-
ing—review & editing.
Funding This research did not receive any specific grant from funding
agencies in the public, commercial, or nonprofit sectors.
Data Availability The nucleotide sequence data reported here are avail-
able in the GenBank database under the accession numbers OL744209-
OL744220 (phage genomes), and OP492053-OP492074 (Bacteria 16S
rRNA seq).
Code Availability Not applicable.
Declarations
Conflict of interest Disclosure of potential conflict of interests: The
authors declare that they do not have any conflict of interests or per-
sonal competing that could influence the work reported in this paper.
Ethical Approval Not applicable.
Informed consent to participate Not applicable.
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