1 Non-Lactose Fermenting (NLF) Escherichia coli: Following In The Footsteps

2 of Lactose Fermenting E. coli High-risk Clones

3

4 Razib Mazumder1, Jody Phelan2, Susana Campino2, Arefeen Haider1, Araf Mahmud1, Dilruba
5 Ahmed3, Taane Clark2, Dinesh Mondal1.
1
6 Laboratory Sciences and Services Division, International Centre for Diarrhoeal Disease Re-
7 search (icddr,b), Bangladesh, Dhaka, Bangladesh
2
8 London School of Hygiene and Tropical Medicine, London WC1H 9SH, UK
3
9 Clinical Microbiology and Immunology Laboratory, Laboratory Sciences and Services Divi-
10 sion, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh

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16 * Address correspondence to:

17 Dr. Dinesh Mondal
18 Acting Senior Director, Laboratory Sciences and Services Division
19 International Center for Diarrhoeal Diseases Research Bangladesh (icddr,b)
20 Postal address: 68, Shahid Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
21 Telephone number: +880-2-9827001-10, Ext: 3478
22 Facsimile number: +880-2-8812529
23 E-mail address: [email protected]
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29 Abstract

30 Multiresistant pathogenic strains of non-lactose fermenting Escherichia coli (NLF E. coli) are
31 responsible for various intestinal and extraintestinal infections. Although a few studies have
32 characterised such strains to some extent using conventional methods, it has not been
33 comprehensively studied at the genomic level. To address this gap, we used whole-genome
34 sequencing (WGS) coupled with detailed microbiological and biochemical testing to investigate
35 17 NLF E. coli from a diagnostic centre (icddr,b) in Dhaka, Bangladesh. The prevalence of NLF
36 E. coli was 10%, of which 47% (8/17) exhibited MDR phenotypes. All 17 (100%) isolates
37 couldn't ferment lactose sugar and were confirmed as E. coli by API and WGS species
38 identification. WGS- based analysis revealed international high-risk clonal lineages. The most
39 prevalent sequence types (STs) were ST131 (23%), ST1193(18%), ST12 (18%), ST501(12%),
40 ST167(6%), ST 73 (6% and ST12 (6%). Phylogenetic analysis corroborated a striking clonal
41 population among the studied NLF E. coli isolates. The predominant phylogroup detected was
42 B2 (65%). The blaCTX-M-15 ESBL gene was present in 53% of isolates (9/17). A total of 11 out of
43 17 isolates were affiliated with pathogenic pathotypes. All ExPEC pathotypes (100%)
44 demonstrated β-hemolysis. Our study underscores the presence of critical pathogens and MDR
45 clones among NLF E. coli, similar to the lactose fermenting (LF) E. coli. We suggest that NLF
46 E. coli be considered equally capable aslactose fermenting E. coli in causing intestinal and
47 extraintestinal infections. Further, there is a need to undertake systematic, unbiased monitoring
48 of predominant lineages among NLF E. coli that would help in better treatment and prevention
49 strategies.

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57 Introduction

58 Escherichia coli is a versatile Gram-negative bacterium with huge genetic diversity (nia Santos
59 Braz et al.; Mazumder et al., 2021b). It is the most common organism responsible for
60 opportunistic infections. Its primary habitat includes the lower intestinal tract of humans and
61 animals (nia Santos Braz et al.). However, infections with variant strains of E. coli are
62 responsible for various clinical manifestations ranging from diarrhea, urinary tract infections,
63 and life-threatening septicemia, resulting in over two million deaths every year globally (Hussain
64 et al., 2012; Huang et al. Moreover, these infections are often associated with cephalosporin and
65 carbapenem-resistant strains, impacting the mortality of patients and imparting huge health care
66 costs (Logan and Weinstein, 2017)

67 E. coli are nonpathogenic facultative anaerobic flora of the intestinal tract in humans. The gram-
68 negative E. coli bacilli ferments lactose to produce hydrogen sulfide. However, up to 20% of E.
69 coli isolates from patients are reported to be atypical, which are slow or non-lactose fermenters
70 due to the deficiency in enzyme lactose permease encoded by the lacY gene. (Nicoletti et al.,
71 1988; Hossain, 2012; Chang et al., 2014; Yaratha et al., 2017; Johnson et al., 2019). The (NLF)
72 E. coli can be identified as colourless, transparent colonies on MacConkey agar and by negative
73 lactose (sugar) fermentation tests (Hossain, 2012; Siqueira et al., 2021). Various strains of E. coli
74 are equipped to cause different forms of enteric and extraintestinal infections in human hosts
75 with varying propensities (Kaper et al., 2004).The pathogenic strains of E. coli belong to both
76 lactose fermenting and non-lactose fermenting E. coli types. However,little attention is given to
77 these atypical strains of E. coli, particularly the non-lactose fermenting E. coli in routine
78 diagnostic testing laboratories.

79 Escherichia coli as an etiological agent of diarrhea is well established (Colonna et al., 1992;
80 Nataro and Kaper, 1998; Hossain, 2012). Diarrheagenic strains of E. coli fall into different
81 categories possessing distinct pathogenic mechanisms, the three important E. coli strains include
82 enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), and enteroaggregative E.
83 coli (EAEC) (Nataro and Kaper, 1998). These E. coli categories cause a range of clinical
84 syndromes such as watery diarrhea in children, persistent diarrhea, traveller's diarrhea and
85 hemolytic uremic syndrome (Nataro and Kaper, 1998). A few studies have reported that NLF E.

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 86 coli strains were identified as typical pathogenic E. coli isolates, having a possible diarrheagenic
87 role (Nicoletti et al., 1988; Colonna et al., 1992; Hossain, 2012)

88 Urinary tract infections (UTIs) represent one of the most common bacterial infections. It is a
89 serious public health problem affecting around 150 million people worldwide (Flores-Mireles et
90 al., 2015). Escherichia coli is the most common pathogen responsible for complicated and
91 uncomplicated urinary tract infections (Flores-Mireles et al., 2015). Such strains harbour
92 multiple virulence factors which play their role in the pathophysiology of UTIs. These Virulence
93 factors affect bacteria colonisation and evasion of host defences (Hussain et al., 2014; Mazumder
94 et al., 2020a). Recent reports suggest that clonal groups of multiresistant, multivirulentE. coli are
95 increasingly responsible for UTIs, presenting a challenge for treating UTIs (Riley, 2014;
96 Mazumder et al., 2021a). In recent years, a few studies have demonstrated that NLF E. coli have
97 been increasingly isolated from urine specimens in the microbiology laboratories and have
98 reported their clinical significance concerning antibiotic resistance, virulence and emerging
99 clones (Chang et al., 2014; Chakraborty et al., 2016; Wu et al., 2017)

100 Rapid identification of bacterial pathogens in microbiology laboratories is critical for initiating
101 successful infection treatment. Screening of gram-negative bacteria from urine and stool samples
102 is routinely performed on MacConkey agar, and the colourless transparent non-lactose
103 fermenting (NLF) E. coli variants are usually missed in the screening process as little attention is
104 given to these atypical strains of E. coli. Although reports of such isolates are limited worldwide,
105 a few studies have reported the occurrence and characterisation of NLF E. coli from clinical
106 specimens. They have characterised the strains using only conventional methods. This study
107 aims to decipher the biochemical profiles, population structure, and genomic characteristics of
108 NLF E. coli from stool and urine samples isolated at a referral diagnostic centre (icddr,b) in
109 Dhaka, Bangladesh. Studies such as these will inform us about the evolutionary trajectories of
110 atypical E. coli variants and shed light on their clinical and public health significance.

111
112 Materials and methods

113 Bacterial isolates

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114 E. coli (175 isolates) was isolated from urine (98 isolates) and stool (77 isolates) culture plates.
115 The isolates were collected randomly from the Clinical Microbiology Laboratory of the
116 International Center for Diarrheal Disease Research, Bangladesh (icddr,b), located in Dhaka,
117 Bangladesh. The isolates were sampled between 2019 and 2020 as part of a larger study on 1%
118 AMR surveillance. From this collection of 98 urine and 77 stool E. coli isolates, we identified 9
119 and 8 NLF E. coli isolates, respectively. These 17 NLF E. coli were subjected to complete
120 biochemical profiling and whole-genome sequencing. NLF E. coli were presumptively identified
121 by their inability to ferment lactose on the MacConkey agar. Further, confirmation was made by
122 standard biochemical methods, sugar fermentation tests and API 20E (Table 1). Hemolytic
123 properties of strains were evaluated using 5% sheep blood agar plates. Clearing zones around the
124 colonies were suggestive of beta-hemolysis.

125 Antimicrobial susceptibility testing

126 The Genome Centre at icddr,b reassessed 17 NLF E. coli isolates with an extensive panel of 18
127 antibiotics (Oxoid, US) by the Kirby-Bauer disk diffusion method. The results were interpreted
128 by referring to the 2019 CLSI guidelines. Upon AST the NLF E. coli isolates identified as
129 intermediate and resistant were considered as resistant. They were classified as MDR if they
130 were nonsusceptible to at least one agent belonging to three different antimicrobial classes.

131 Whole-genome sequencing

132 Total bacterial DNA was isolated and purified from the overnight grown bacterial cultures using
133 the QIAmp DNA Mini kit (Qiagen, Germany). DNA QC and quantification were performed
134 employing a Nanopore spectrophotometer (Thermo Fisher Scientific, United States) and
135 Qubit4.0 fluorometer (Life Technologies) (Baddam et al., 2020). DNA libraries for the short-
136 read paired-end sequencing were prepared using the Nextera DNA Flex library prep kit
137 (Illumina). Size selected and pooled libraries were sequenced at the icddr,b Genome Centre in
138 Illumina NextSeq 500 system using a NextSeq v2.5 reagent kit (2 × 150 bp) (Mazumder et al.,
139 2020b).

140 Sequence assembly and annotation

141 High-quality reads were obtained by filtering the paired-end data using fastp(Chen et al.). De
142 novo assemblies were generated using SPAdes v.3.11.11(Bankevich et al., 2012). NCBI

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143 Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016) was used for
144 annotating the genome assemblies. The genome statistics from the resulting file were gleaned
145 using quast 5.2.0 (Gurevich et al., 2013)

146 In silico analysis

147 Species identification was confirmed using Kmer Finder version 3.2 (Hasman et al., 2014;
148 Larsen et al., 2014; Clausen et al., 2018). Phylogenetic groups of the genomes were inferred
149 using the Clermon Typing tool (Beghain et al., 2018). The sequence types (STs) were
150 determined by submitting the contigs to MLST version 2.0, scheme #1 (Larsen et al., 2012).
151 SerotypeFinder 2.0 (Joensen et al., 2015) and CH typer 1.0 (Roer et al., 2018) were used for
152 elucidating serotypes and clonotypes, respectively.

153 Resistance genes were screened by BLASTn analysis of the genome assemblies against the data
154 downloaded from the ResFinder database (Bortolaia et al., 2020). The 70% query coverage and
155 90% identity indicated a positive hit in the genome. Plasmid incompatibility groups were
156 determined by PlasmidFinder 2.1 (Carattoli et al., 2014). Mobile genetic elements associated
157 with acquired AMR genes were determined using Mobile Element finder v1.0.3 (Johansson et
158 al., 2021). Chromosomal point mutations conferring fluoroquinolone resistance were identified
159 by PointFinder(Zankari et al., 2017). Contigs with blaNDM-5 and blaCTX-M-15 were analysed by
160 BLASTn analysis against the database in NCBI to identify the chromosomal or plasmid origin of
161 these genes.

162 Virulence gene content of strains was determined by BLASTn analysis of 17 genomes against
163 the E. coli database selected in VFDB (Chen et al., 2005). The genes with 90% identity and 70%
164 query coverage were considered present. Isolates were classified as ExPEC based on Johnson's
165 criteria (Johnson and Stell, 2000)

166 The In-silico analysis described above was performed on default parameters unless otherwise
167 stated.

168 Phylogenomic and pangenomic analysis

169 The phylogenetic tree was inferred with the reference sequence alignment-based phylogeny
170 builder (REALPHY) (Bertels et al., 2014) using the default 50-readlength parameter.E. coli K-12

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171 MG1655 genome(accession No.U00096) was used as the single reference sequence and PhyML
172 (Guindon et al., 2010) was used as the treebuilder. The phylogenetic tree was later visualised
173 using the Interactive Tree of Life (iTOL)(Letunic and Bork, 2016). Pangenomic analysis was
174 conducted using the Anvi'o 7.1(Eren et al., 2015) software package following the microbial
175 pangenomics analysis workflow.

176 Data availability

177 The whole-genome sequence data of the 17 studied NLF E. coli isolates were deposited in NCBI
178 (GenBank) under the BioProject identifier (ID) PRJNA839767. The accession numbers of all 17
179 genomes are enlisted in Table 2.

180 Results

181 Bacterial and biochemical characteristics

182 The colourless non-lactose fermenting (NLF) colonies on the MacConkey agar plate appeared
183 like Shigellaspp colonies. Colonies were colourless (NLF) even in 24 hr old MacConkey agar
184 cultures, removing the possibility of late lactose fermenters. Serology of all colonies revealed
185 negative reaction to the four serogroups of Shigella: S. flexneri, S. dysenteriae, S. boydii, and S.
186 sonnei. Further, streaking of colonies on CHROMagar Orientation media revealed small, pink-
187 red colonies typical of E. coli. The isolates were then tested by routine biochemical tests
188 described in table 1 and identified as E. coli. Notably, all isolates were ONPG positive and did
189 not ferment lactose sugar. Further confirmation was done by generating a biochemical profile for
190 each isolate using 20 reactions of the API 20E strip. The API results gave three distinct API
191 profiles: 7144532 (n=11), 5044552 (n=3) and 5144572 (n=3) and identified all isolates as E. coli
192 with 99.8% probability (Table 1). Gram-staining confirmed gram-negative bacilli. The optimum
193 growth temperate of NLF E. coli isolates ranged from 26°-42°C.

194 Molecular analysis and phylogenomics of NLF E. coli isolates

195 WGS of the 17 NLF E. coli study isolates yielded an average genome size of 5,160,336 bp
196 (range 4,890,228 to 5,480,336 and the average GC content was 50.6% (range 50.3 to 50.7).
197 KmerFinder results confirmed all the isolates as E. coli. On average, the genome assemblies had
198 a genome coverage of 86X (range from 62 X to 124X) (Table 2).

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199 According to in silico MLST, isolates were classified into nine distinct sequence types (STs) and
200 they represented some of the international high-risk lineages as shown in Fig. 1. ST131 was the
201 predominant ST that accounted for 4 (23%) of the 17 NLF E. coli isolates. ST1193 and ST12
202 were present in equal proportions, 3 (18%) each, followed by ST501 2 (12%). Other significant
203 STs detected were ST167 (6%), ST73 (6%) and ST12 (6%). The predominant phylogroup
204 detected was B2 (65%) which comprised all isolates affiliated with ST131, ST1193, ST12 and
205 ST73. This was followed by group A (n=2: 1ST167; 1 ST2089); other phylogroups were
206 represented by single isolates (Fig. 1). By serogroup, the 4 ST131 isolates were either O16:H4
207 (n=2: fumC40:fimH41) or O25:H4 (n=2: fumC40:fimH30) (Fig. 1). All ST1193 isolates were
208 O75:H5 with fumC14:fimH64. All ST12 isolates were O4:H1and had fumC13. Other STs
209 exhibited diverse serotypes and CH types (Fig. 1)

210 Based on the alignment of detected core genome SNPs, a phylogenetic tree was built for 17 NLF
211 E. coli from our study with the MG1655 genome as the reference genome. There was relatively
212 low diversity of the studied NLF E. coli as the majority of strains clustered together, resulting in
213 clades and subclades. The analysis clustered the 17 isolates into two major clades, one tight
214 cluster corresponding to phylogroup B2 and the other loosely clustered clade included strains
215 from A, B1, D and F phylogroup. Compared to NLF E. coli from stool, the urine E. coli isolates
216 formed a tight clade with strains belonging to ST12, ST1193 and ST131. Three strains from stool
217 origin clustered closely with urine strains reinforcing the gut as a possible reservoir of UTI-
218 causing agents. The blaCTX-M-15, MDR phenotype and β-hemolysis did not correspond with the
219 clusters indicating their widespread presence across E. coli lineages. However, the sequence
220 types, serogroups, and CH-types corresponded closely with the phylogenetic clusters of E. coli
221 genomes. The ST131 strains with O16:H5 (fumC40:fimH41) formed a subcluster with ST131
222 strains having O25:H4 (fumc40:fimH30), indicating there are sublineages within ST131.

223 We also analysed the pangenomes of the 17 NLF E. coli genomes (Fig. 2). The number of core
224 genes identified was 3400, and the accessory genes were 30563. Contrary to the core-genome-
225 based phylogenetic tree, no two genomes shared the same gene content with respect to the
226 pangenome. A closer look into the pangenome dendrogram reflects a clustering pattern that
227 mimics the clades of the core genome-based phylogenetic tree (Fig. 2). These observations

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228 indicate that the 17 NLF E. coli genomes do not differ drastically, even with regard to their
229 accessory gene content. They all have a few unique gene families, as shown in figure 2.

230 Virulence genes

231 Our analysis identified 73 virulence-associated genes out of 335 genes analysed. The distribution
232 of 73 virulence genes among 17 NLF E. coli isolates is shown in Fig. 3. Overall, 65% (11/17) of
233 all NLF E. coli isolates were assigned to different pathotypes, as classified by the presence of
234 specific virulence markers. The majority of urine NLF E. coli isolates were assigned to the
235 EXPEC pathotype [67% (6/9)]. Suspected diarrheagenic variants comprised 62% (5/8) of the
236 stool NLF E. coli isolates. The most prevalent pathotype among diarrheagenic variants was
237 enteroaggregative E. coli (EAEC) (38%) which harboured both aggR and aatA genes, followed
238 by enteropathogenic E. coli (EPEC) (13%) that showed the presence of intimin gene (eae). One
239 isolate from stool origin was also classified as ExPEC. The remaining six isolates (6/17) that did
240 not belong to any pathotype included three strains from ST1193, two from ST131 and one from
241 ST501. Out of 4 ST131 studied strains, two O25:H4 serogroup (fimH30) strains qualified as
242 ExPEC and two strains with O16:H5 serogroup (fimH41) did not qualify as ExPEC. Among all
243 the pathotypes, the seven (100%) ExpEC strains produced β-hemolysis in 5% sheep blood agar.
244 This hemolytic phenotype was strongly associated with alpha-hemolysin genes (hlyA, hlyB and
245 hlyD). Although the three ST1193 isolates did not qualify as ExpEC pathotypes, they all carried
246 a similar set of virulence genes, including adhesins (fimH), toxins (sat, hlyE/clyA), siderophores
247 (chuA, fyuA, sitABCD, iutA) and others. The NLF E. coli belonging to urine origin had a higher
248 prevalence of virulence genes as the aggregate virulence score [median (range)] for urine isolates
249 [90 (72-114)] was higher than that of stool NLF E. coli isolates [73 (62-116)].

250 Antimicrobial resistance

251 The phenotypic and genotypic trends of antimicrobial resistance of the studied NLF E.
252 coli strains are shown in Fig. 4. Overall, the NLF E. coli strains from the stool and urine samples
253 showed high antimicrobial drug resistance rates to nalidixic acid (100%), ceftazidime (94%),
254 cefradine (94%), followed by ciprofloxacin (88%), ampicillin (82%), cefotaxime (65%) and
255 cefepime (65%). The resistance rates to cefuroxime (59%), ceftriaxone (53%), co-trimoxazole
256 (41%), imipenem (35%) were moderate and the resistance rates to gentamicin (18%), amikacin
257 (12%), doxycycline (6%), meropenem (6%) were low. In contrast, the resistance rate to

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258 chloramphenicol, fosfomycin and tigecycline was 0. Compared to the stool isolates, the urine
259 NLF E. coli isolates had relatively low resistance to amikacin, cefepime, ceftriaxone and
260 cefuroxime; consequently, there were 63% (5/8) MDR isolates in the stool category and 33%
261 (3/9) MDR isolates in urine category.

262 We detected 31 unique AMR gene alleles belonging to different classes, with most genomes
263 exhibiting at least 6 AMR genes (Fig. 4). By clinical source, the AMR genes had a relatively
264 similar distribution between the stool and urine NLF E. coli isolates, with strains from both the
265 groups having a minimum of 6 AMR genes per genome, albeit substantial variations among
266 isolates (range 3-15 for stool E. coli and 2-13 for urine E. coli isolates). No specific AMR profile
267 was associated with isolates from a particular clinical source.

268 A variety of ESBL gene variants encoded cephalosporin resistance. Amongst the identified
269 ESBL genes in NLF E. coli isolates, blaCTX-M-15, was predominant [9/17 (53%)], but we also
270 identified blaOXA-1, blaTEM1B, blaDHA-1 and blaTEM-1C genes in a number of isolates (Fig. 4). We
271 observed 100% concordance between the presence of blaCTX-M-15 gene and resistance phenotypes
272 to the following cephalosporin antibiotics, cefradine, cefotaxime, ceftazidime, ceftriaxone,
273 cefuroxime and cefepime. One blaCTX-M-15 positive stool NLF E. coli harboured a blaNDM-1 gene;
274 this strain (A4) demonstrated difficult-to-treat resistance (DTR) as it was resistant to 14 of 18
275 antibiotics tested, including all first-line agents. It was sensitive to chloramphenicol,
276 doxycycline, fosfomycin and tigecycline. The probable genome locus of blaCTX-M-15 gene was
277 detected to be a plasmid for 4 out of 9 NLF E. coli isolates, as shown in Table 3. The genetic
278 environment of blaCTX-M-15 gene in a majority of isolates [56% (5/9)] consisted of the insertion
279 element ISEcp1preceeding the gene (Table 3). The blaCTX-M-15 gene was mainly associated with
280 the epidemiological significant clonal E. coli lineages such as ST131, ST167, ST1193 and
281 ST6303. Interestingly, these strains were all non-lactose fermenting (NLF).

282 We screened for mutations in the quinolone-resistance-determining regions (QRDRs) of gyrA,

283 parC and parE. We identified amino acid substitutions at codon positions S83I (16/17 isolates),
284 D87N (9/17 isolates) in the amino acid sequence of gyrA and S80I (9/17 isolates), S57T (one
285 isolate), E84V (4/17 isolates), E84G (two isolates) and S80R (two isolates) in parC. We also
286 identified mutations at codon positions S45A, I529L and L416F in parE gene. The gyrA
287 mutation S83L was strongly associated with resistance to ciprofloxacin. The ESBL gene blaCTX-M-

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288 15 and gyrA S83L, parC E84V were strongly linked in ST131 and ST167 lineage strains.
289 Additionally, the isolated also harboured acquired AMR genes encoding fluoroquinolone (FQR)
290 resistance, including Qnrs1 (one isolate), Qnrs13 (one isolate) and aac(6')-Ib-cr gene (3
291 isolates). All the three acquired FQR genes were associated with ciprofloxacin resistance.

292 Multidrug resistance, defined by resistance to a minimum of one agent belonging to three
293 different antimicrobial classes, was frequently observed in isolates with multiple AMR genes
294 identified in 8 (47%) isolates. Cephalosporin resistance genes and mutation in gyrA S83L were
295 strongly associated with the MDR phenotype.

296 PlasmidFinder identified seven unique plasmid replicon groups, with each isolate carrying an
297 average of 2.5 plasmid groups (Fig.3). FIB was the most commonly encountered plasmid group
298 (n=13/17), followed by FII (n=9/17), CoI (n=8/12) and FIA (n=7/17). FIA, FIB and CoI
299 plasmids were strongly associated with ST131 E. coli. The NDM-positive E. coli strain (A4)
300 concomitantly hosted three IncF-type replicons, FIA, FIB, and FII groups. We could not
301 associate AMR genes with known plasmid replicons in two strains (A6 & U8) as no replicons
302 were detected in them. Surprisingly, these strains belong to well-known ESBL-producing
303 lineages (ST501 and ST131, respectively).

304

305 Discussion

306 We found a high prevalence of epidemiologically significant clonal lineages among NLF E. coli
307 isolates from patients with suspected UTIs and enteric infections in a referral diagnostic centre
308 (icddr,b) in Dhaka, Bangladesh. None of the previous studies has focused on the genomic
309 epidemiology of NLF E. coli. In the current study, we carried out a comprehensive
310 characterisation of 17 NLF E. coli isolates to delineate their microbiological, biochemical and
311 molecular characterisation using WGS to identify their population composition and detect their
312 molecular markers of AMR and virulence. This first report forms a baseline study on the
313 genomic epidemiology of clinical NLF E. coli isolates. Knowledge about such strains' virulence
314 and resistance properties is important to help direct researchers, clinicians and laboratory
315 technologists screen, analyse and report atypical variants of E. coli recovered from clinical
316 samples.

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317 In line with previous reports (Hossain, 2012; Yaratha et al., 2017), our study revealed that the
318 prevalence rate of NLF E. coliin patients in the community is around 10%. This makes it
319 important to identify NLF E. coli in clinical specimens, which would help initiate appropriate
320 antimicrobial therapy. Given the difficulty in differentiating between NLF E. coli and Shigella
321 spp, multiple tests need to be performed as described in this study, including serology,
322 biochemical identification (manual and API) and molecular methods like WGS. It is reported
323 that routine MALDI-TOF MS analysis cannot reliably differentiate between E. coli and Shigella
324 species (Ling et al., 2019). It can be noted here that the chromogenic media- CHROMagar
325 Orientation cannot distinguish between lactose fermenting and non-lactose fermenting E. coli
326 and can be used to isolate/identify both types of E. coli in one go.

327 The predominant sequence types detected in this study were epidemiologically significant high-
328 risk clones, including ST131, ST1193, ST12, ST73 and ST167. Our group previously reported
329 these clones from Bangladesh among ESBL-producing lactose fermenting E. coli(Mazumder et
330 al., 2021a). None of the studies has reported them from NLF E. coli except for ST1193 (Wu et
331 al., 2017). The dominance of clonal sequence types among this collection of NLF E. coli is
332 interesting. It warrants close attention because the inclusion of isolates in this study was not
333 based on any antimicrobial resistance phenotypes and genotypes. These STs are predominantly
334 reported to be associated with extraintestinal infections (Pitout, 2012; Riley, 2014; Manges et al.,
335 2019). Also, a review article on global ExPEC STs enlisted these STs to be responsible for the
336 enormous burden of extraintestinal human infections globally (Manges et al., 2019). These
337 highly successful clonal groups are a major mode of spreading antimicrobial resistance by the
338 mechanism of global expansion (Tchesnokova et al.; Shaik et al., 2017).

339 ST131 E. coli is an established pandemic extraintestinal multidrug-resistant pathogen (Rogers et

340 al., 2011; Nicolas-Chanoine et al., 2014). The 4 ST131 isolates identified in our collection were
341 of two O and CH types, two ST131 E. coli exhibited O25:H4 with fumC40:fimH30 and the other
342 two ST131 E. coli had O16:H5 serogroup and fumC40:fimH41 CH type. The two strains with
343 O25: H4 serogroup belonged to the subclade C2, known as the H30Rx sublineage, which is the
344 highly drug-resistant and successful sublineage of the ST131 clone. ST1193 is reported to be an
345 emerging multiresistant clonal group (Johnson et al., 2019). The three ST1193 isolates in this
346 study carried the same set of three nonsynonymous chromosomal mutations in gyrA (S83L),

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347 gyrA (D87N) and parC (S80I) that confer fluoroquinolone resistance and had O75:H5 serogroup
348 with fumC14: fimH64 CH type, they were all ciprofloxacin-resistant. ST1193 prevalence is
349 increasing rapidly worldwide and is expected to replace the most successful clone, ST131, in the
350 near future (Pitout et al., 2022). The three ST12 isolates in this collection were qualified as
351 ExPEC pathogens and were ciprofloxacin-resistant. ST12 is reported to be one of the
352 predominant STs that cause bloodstream infections (Kallonen et al., 2017; Manges et al., 2019).
353 The ST73 isolate detected in this study harboured 104 of 335 virulence genes, including several
354 markers of ExPEC and sepsis-associated E. coli (SEPEC). In contrast, it carried a smaller
355 number of AMR genes. It demonstrated β-hemolysis on 5% sheep blood agar and had the
356 CHtype-fumC24:fimH30. THE ST167 strain we identified showed a difficult-to-treat phenotype
357 and carried blaNDM-5 gene. It qualified as an EAEC pathotype and this strain has been associated
358 with a global spread of ESBL-E. coli in humans (Ewers et al., 2012). ST167 associated with
359 blaNDM-5 gene was reported as an emerging carbapenem-resistant high-risk clone (Garcia-
360 Fernandez et al., 2020).

361 Overall, the NLF E. coli isolates carried the AMR genes and were moderately resistant to
362 antibiotics, but a higher number were assigned to different pathotypes. The isolates carried
363 extensive virulence genes that belonged to different categories, including adhesion, invasion,
364 colonisation and iron uptake ability. Nevertheless, the combination of resistance and virulence in
365 these NLF E. coli isolates could contribute to their increased fitness and potential to expand
366 globally. This study has multiple limitations. First, limited clinical and epidemiological data
367 were available. Second, a small sample size constrained the power to generalise our findings and
368 confirm statistical associations, particularly with the high-risk clones. The main gain of the study
369 is that emphasis on inclusion was not laid on MDR or ESBL phenotypes; this has led to a clear
370 understanding of the important ExPEC lineages within NLF E. coli. The WGS approach helped
371 to provide a greater resolution to the study observations.

372 In conclusion, the prevalence of NLF E. coli variants isolated at a referral diagnostic centre
373 (icddr,b) in Dhaka, Bangladesh, was moderate (10%). Our study revealed multiresistant strains
374 and high-risk clonal groups, including ST131, ST1193, ST12, ST73 and ST167, emerging within
375 NLF E. coli isolates. To our knowledge, this is the first report on such a phenomenon in NLF E.
376 coli; most studies focus on pathotypes encompassing single STs. These high-risk clones may

13 | P a g e
377 further evolve by acquiring carbapenem and colistin resistance genes to cause difficult-to-treat
378 infections. Therefore, strengthening microbiology laboratories to detect and report NLF E. coli
379 isolates is important to accomplish successful patient treatment and to feed data to AMR
380 surveillance programs. Further national and regional multicentre One Health studies are required
381 to ascertain the significance of NLF E. coli as significant pathogens impacting public health.

382 Acknowledgments: This research study was funded by core donors which provide unrestricted
383 support to icddr,b for its operations and research. Current donors providing unrestricted support
384 include: Government of the People's Republic of Bangladesh; Global Affairs Canada (GAC);
385 Swedish International Development Cooperation Agency (Sida) and the Department for
386 International Development (UK Aid). We gratefully acknowledge these donors for their support
387 and commitment to icddr,b's research efforts.
388
389 Transparency declarations: None to declare
390

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561

562

563

564

565

566

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567 Figure Legends
568 Figure 1. Phylogenetic relationships amongst sequenced NLF E. coli genomes. The
569 maximum likelihood phylogenetic tree is based on the alignment of detected core genome SNPs
570 of 17 NLF E. coli genomes with the MG1655 genome strain as a reference genome. The
571 phylogroups, clinical source, blaCTX-M-15 status, beta-hemolysis, MDR status, STs, serogroups, CH
572 types and blaNDM-5 status are shown to the right of the tree.
573

574 Figure 2.
575 Pangenome analysis of 17 NLF E. coli genomes from Anvi'o pangenomics suite. The circles
576 represent individual genomes arranged as per their pangenomic phylogenetic relationship
577 depicted by the outside dendrogram. In the circles, dark colour indicates the presence of a gene
578 group and light colour its absence.
579

580 Figure 3.
581 Heat map depicting the distribution of 73 virulence genes among 17 NLF E. coli genomes. Dark
582 brown represents the presence, and light brown blocks represent a virulence gene's absence.
583

584 Figure 4.
585 Heat map demonstrating the AST profile, plasmid replicon types and acquired AMR gene profile
586 of 17 NLF E. coli isolates/genomes. Dark green represents resistance/ presence and light green
587 blocks represent the sensitivity/ absence of a particular trait. Definition of abbreviated antibiotics
588 for AST: AMK, amikacin; Amp, ampicillin; FEP, cefepime; CTX, cefotaxime; CAZ,
589 ceftazidime; RAD, cephradine; CRO, ceftriaxone; CXM, cefuroxime; CHL, chloramphenicol;
590 CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole; DOX, doxycycline; FOF, fosfomycin;
591 GEN, gentamicin; IPM, imipenem; MEM, meropenem; NAL, nalidixic acid; TGC, tigecycline.
592

593

594

595

596

597

598

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599 Table 1: Microbiological and biochemical characteristics of 17 NLF E. coli isolates

No. (%)
Sl. No. Biochemical tests
Positive Negative
1 Catalase test 17 (100%) 0
2 Oxidase test 0 17 (100%)
3 TSI agar
a. Acid production in slant 17 (100%) 0
b. Acid production in butt 17 (100%) 0
c. Hydrogen sulfide production(H2S) 0 17 (100%)
d. Gas production 17 (100%) 0
4 Motility Indole Urea's Test (MIU)
a. Motility 17 (100%) 0
b. Indole Production 17 (100%) 0
c. Urea hydrolysis 0 17 (100%)
5 Simmons Citrate reaction test 0 17 (100%)
6 Acetate 17 (100%) 0
7 Sugar fermentation
a. Glucose 17 (100%) 0
b. Lactose 0 17 (100%)
c. Sucrose 11 (65%) 6(35%)
e. Mannose 17 (100%) 0
f. Arabinose 17 (100%) 0
g. Sorbitol 17 (100%) 0
h. Mannitol 17 (100%) 0
i. Inositol 0 17 (100%)
8 Ortho-Nitrophenyl-β-galactoside (ONPG) 17 (100%) 0
9 Gelatin liquefaction 0 17 (100%)
10 Vogas-proskauer 0 17 (100%)
11 Lysine decarboxylase 17 (100%) 0
12 Ornithine decarboxylase 14(82%) 3 (18%)
13 Arginine Dihydrolase 11 (65%) 6(35%)
API 20E Results [API profile: isolates]
1 API profile: 7144532 A3, A4, A5, A7, U1, U7, U8, U12, U13, U14, U15
2 API profile: 5144572 A6, U4, U6
3 API profile: 5044552 A1, A8, A9
Bacteriological Characteristics [17 (100%)]
1 Growth on MacConkey Agar Colorless and transparent (NLF colony)
2 Growth on SS agar Colorless and transparent (NLF colony)
3 Growth on CHROMagar™ Orientation Pink color colony
4 Growth Temperature (26-42°C)
5 Gram staining Negative rods
600

601

602

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603 Table 2: Genomic features of the 17 whole-genome sequenced NLF E. coli isolates
Geno Contig
Isol Isolati
Sl Strain me No. Genome No. GC
ate Source on Accession No.
No. ID covera (>500 size (bp) CDS %
ID year
ge bp)
BDEc_N JAMKCS00000
1 A1 LF-A1 Stool 2019 91 106 5,305,815 5077 50.5 0000
BDEc_N JAMKCT00000
2 A3 LF-A3 Stool 2019 101 103 5,480,336 5335 50.6 0000
BDEc_N JAMKCU00000
3 A4 LF-A4 Stool 2019 96 96 5,019,286 4772 50.7 0000
BDEc_N JAMKCV00000
4 A5 LF-A5 Stool 2019 63 77 5,074,445 4830 50.5 0000
BDEc_N JAMKCW0000
5 A6 LF-A6 Stool 2019 121 84 4,890,228 4656 50.6 00000
BDEc_N JAMKCX00000
6 A7 LF-A7 Stool 2020 90 70 4,978,296 4710 50.7 0000
BDEc_N JAMKCY00000
7 A8 LF-A8 Stool 2020 124 151 5,033,902 4934 50.6 0000
BDEc_N JAMKCZ00000
8 A9 LF-A9 Stool 2020 91 43 5,068,010 4850 50.6 0000
BDEc_N JAMKDA00000
9 U-1 LF-U1 Urine 2019 71 44 5,073,930 4893 50.6 0000
BDEc_N JAMKDF00000
10 U-4 LF-U4 Urine 2019 62 53 5,251,806 4988 50.4 0000
BDEc_N JAMKDG00000
11 U-6 LF-U6 Urine 2019 79 44 5,054,491 4847 50.6 0000
BDEc_N JAMKDH00000
12 U-7 LF-U7 Urine 2019 88 109 5,302,191 5061 50.5 0000
BDEc_N JAMKDI00000
13 U-8 LF-U8 Urine 2020 71 56 5,186,272 5002 50.6 0000
U- BDEc_N JAMKDB00000
14 12 LF-U12 Urine 2020 83 57 5,021,789 4793 50.7 0000
U- BDEc_N JAMKDC00000
15 13 LF-U13 Urine 2020 84 69 5,262,816 5008 50.5 0000
U- BDEc_N JAMKDD00000
16 14 LF-U14 Urine 2020 70 145 5,304,507 5153 50.7 0000
U- BDEc_N JAMKDE00000
17 15 LF-U15 Urine 2020 75 79 5,417,595 5190 50.36 0000
604
605
606
607
608
609
610
611

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612 Table 3: Characteristics of CTX-M-15 associated NLF E. coli isolates

Isolate Genome
ID Source ST Phylogroup locus MGEsa Plasmid replicon
A1 Stool 6303 D Plasmid ISEcp1 IncFIB, IncFII
A3 Stool 131 B2 Plasmid NDb Col, IncFIA, IncFIB, IncFII, IncY
A4 Stool 167 A Chromosome ISEcp1 IncFIA, IncFIB, IncFII
A5 Stool 501 D Chromosome ISEcp1 IncFIB, IncFII
A7 Stool 131 B2 Chromosome NDb Col, IncFIA, IncFIB, IncFII
A8 Stool 2089 A Plasmid ISKpn19 IncFIB
A9 Stool 1193 B2 Chromosome ISEcp1 Col, IncFIA
U-8 Urine 131 B2 Plasmid IS3 NDb
U-12 Urine 131 B2 Chromosome ISEcp1 Col, IncFIA, IncFIB, IncL

614 a
Mobile genetic elements
615 b
Not detected
616

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